Peptide nucleic acids having 2,6-diaminopurine nucleobases

ABSTRACT

A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. The peptide nucleic acids of the invention comprise ligands selected from a group consisting of naturally-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone. Some PNAs of the invention also contain C 1 -C 8  alkylamine side chains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/847,110, filed May 1, 1997, now abandoned, which is a divisional ofSer. No. 08/686,114, filed Jul. 24, 1996 now U.S. Pat. No. 6,414,112,which is a continuation-in-part of U.S. application Ser. No. 08/108,591,filed Nov. 22, 1993 now U.S. Pat. No. 6,395,474. Application Ser. No.08/108,591, in turn, derives from Danish Patent Application No. 986/91,filed May 24, 1991, Danish Patent Application No. 987/91, filed May 24,1991, and Danish Patent Application No. 510/92, filed Apr. 15, 1992. Theentire disclosure of each of the above-mentioned is incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention is directed to peptide nucleic acids (PNAs)wherein naturally-occurring nucleobases or non-naturally-occurringnucleobases are covalently bound to a polyamide backbone. One preferrednaturally-occurring nucleobase is 2,6-dimaminopurine. Some PNAs of thepresent invention comprise at least one 2,6-diaminopurine nucleobasewhile other PNAs comprise at least one 2,6-diaminopurine nucleobase andat least one C₁-C₈ alkylamine side chain. The PNAs of the invention bindDNA with enhanced binding affinity and exhibit higher sequencespecificity.

BACKGROUND OF THE INVENTION

The function of a gene starts by transcription of its information to amessenger RNA (mRNA). By interacting with the ribosomal complex, mRNAdirects synthesis of the protein. This protein synthesis process isknown as translation. Translation requires the presence of variouscofactors, building blocks, amino acids and transfer RNAs (tRNAs), allof which are present in normal cells.

Most conventional drugs exert their effect by interacting with andmodulating one or more targeted endogenous proteins, e.g., enzymes.Typically, however, such drugs are not specific for targeted proteinsbut interact with other proteins as well. Thus, use of a relativelylarge dose of drug is necessary to effectively modulate the action of aparticular protein. If the modulation of a protein activity could beachieved by interaction with or inactivation of mRNA, a dramaticreduction in the amount of drug necessary, and the side-effects of thedrug, could be achieved. Further reductions in the amount of drugnecessary and the side-effects could be obtained if such interaction issite-specific. Since a functioning gene continually produces mRNA, itwould be even more advantageous if gene transcription could be arrestedin its entirety. Oligonucleotides and their analogs have been developedand used as diagnostics, therapeutics and research reagents. One exampleof a modification to oligonucleotides is labeling with non-isotopiclabels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, orother reporter molecules. Other modifications have been made to theribose phosphate backbone to increase the resistance to nucleases. Thesemodifications include use of linkages such as methyl phosphonates,phosphorothioates and phosphorodithioates, and 2′-O-methyl ribose sugarmoieties. Other oligonucleotide modifications include those made tomodulate uptake and cellular distribution. Phosphorothioateoligonucleotides are presently being used as antisense agents in humanclinical trials for the treatment of various disease states. Althoughsome improvements in diagnostic and therapeutic uses have been realizedwith these oligonucleotide modifications, there exists an ongoing demandfor improved oligonucleotide analogs.

In the art, there are several known nucleic acid analogs havingnucleobases bound to backbones other than the naturally-occurringribonucleic acids or deoxyribonucleic acids. These nucleic acid analogshave the ability to bind to nucleic acids with complementary nucleobasesequences. Among these, the peptide nucleic acids (PNAs), as described,for example, in WO 92/20702, have been shown to be useful as therapeuticand diagnostic reagents. This may be due to their generally higheraffinity for complementary nucleobase sequence than the correspondingwild-type nucleic acids.

PNAs are compounds that are analogous to oligonucleotides, but differ incomposition. In PNAs, the deoxyribose backbone of oligonucleotide isreplaced by a peptide backbone. Each subunit of the peptide backbone isattached to a naturally-occurring or non-naturally-occurring nucleobase.One such peptide backbone is constructed of repeating units ofN-(2-aminoethyl)glycine linked through amide bonds.

PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. Theresulting PNA/DNA or PNA/RNA duplexes are bound tighter thancorresponding DNA/DNA or DNA/RNA duplexes as evidenced by their highermelting temperatures (T_(m)). This high thermal stability ofPNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNAbackbone, which results elimination of charge repulsion that is presentin DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA)duplexes is that T_(m) is practically independent of salt concentration.DNA/DNA duplexes, on the other hand, are highly dependent on the ionicstrength.

Homopyrimidine PNAs have been shown to bind complementary DNA or RNAforming (PNA)₂/DNA(RNA) triplexes of high thermal stability (Egholm etal., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992,114, 1895; Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677).

In addition to increased affinity, PNAs have increased specificity forDNA binding. Thus, a PNA/DNA duplex mismatch show 8 to 20° C. drop inthe T_(m) relative to the DNA/DNA duplex. This decrease in T_(m) is notobserved with the corresponding DNA/DNA duplex mismatch (Egholm et al.,Nature 1993, 365, 566).

A further advantage of PNAs, compared to oligonucleotides, is that thepolyamide backbone of PNAs is resistant to degradation by enzymes.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs that bind to complementaryDNA and RNA strands for use as diagnostics, research reagents andpotential therapeutics. For many applications, the oligonucleotides andoligonucleotide analogs must be transported across cell membranes ortaken up by cells to express their activity.

PCT/EP/01219 describes novel PNAs which bind to complementary DNA andRNA more tightly than the corresponding DNA. It is desirable to appendgroups to these PNAs which will modulate their activity, modify theirmembrane permeability or increase their cellular uptake property. Onemethod for increasing amount of cellular uptake property of PNAs is toattach a lipophilic group. U.S. application Ser. No. 117,363, filed Sep.3, 1993, describes several alkylamino functionalities and their use inthe attachment of such pendant groups to oligonucleosides.

U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, and itscorresponding published PCT application WO 94/06815, describe othernovel amine-containing compounds and their incorporation intooligonucleotides for, inter alia, the purposes of enhancing cellularuptake, increasing lipophilicity, causing greater cellular retention andincreasing the distribution of the compound within the cell.

U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993, describesnucleosides and oligonucleosides derivatized to include a thiolalkylfunctionality, through which pendant groups are attached.

Peptide nucleic acids may contain purine or pyrimidine nucleobases.However, previous PNAs having a high purine nucleobase content exhibitdecreased solubility at physiological pH. PNAs of the present inventionovercome this problem.

Despite recent advances, there remains a need for a stable compound thatenhances or modulates binding to nucleic acids, stabilizes thehybridized complexes and increases the aqueous solubility.

SUMMARY OF THE INVENTION

The present invention provides peptide nucleic acids (PNAs) with higherbinding affinity to complementary DNA and RNA than corresponding DNA,The PNAs of the present invention comprise ligands linked to a polyamidebackbone. Representative ligands include the four majornaturally-occurring DNA nucleobases (i.e. thymine, cytosine, adenine andguanine), other naturally-occurring nucleobases (e.g. inosine, uracil,5-methylcytosine, thiouracil or 2,6-diaminopurine) or artificial bases(e.g. bromothymine, azaadenines or azaguanines) attached to a polyamidebackbone through a suitable linker.

The present invention provides a peptide nucleic acid having formula(I):

wherein:

each L is independently selected from a group consisting ofnaturally-occurring nucleobases and non-naturally-occurring nucleobases,at least one of said L being a 2,6-diaminopurine nucleobase;

each R^(7′) is independently hydrogen or C₁-C₈ alkylamine;

R^(h) is OH, NH₂ or NHLysNH₂;

R^(i) is H, COCH₃ or t-butoxycarbonyl; and

n is an integer from 1 to 30.

Preferably, R^(7′) is C₁-C₈ alkylamine. More preferably, R^(7′) is C₃-C₆alkylamine. Even more preferably, R^(7′) is C₄-C₅ alkylamine. Still morepreferably, R^(7′) is butylamine.

Preferably, at least one of the R^(7′) is C₃-C₆ alkylamine. Morepreferably, at least one of the R^(7′) is C₄-C₅ alkylamine. Still morepreferably, at least one of the R^(7′) is butylamine. Even morepreferably, substantially all of the R^(7′) is butylamine.

Preferably, the carbon atom to which substituent R^(7′) are attached isstereochemically enriched. Hereinafter, “stereochemically enriched”means that one stereoisomer is present more than the other stereoisomerin a sufficient amount as to provide a beneficial effect. Preferably,one stereoisomer is present by more than 50%. More preferably, onestereoisomer is present by more than 80%. Even more preferably, onesteroisomer is present by more than 90%. Still more preferably, onestereoisomer is present by more than 95%. Even more preferably, onestereoisomer is present by more than 99%. Still even more preferably,one stereoisomer is present in substantially quantitatively. Preferably,the stereochemical enrichment is of R configuration.

Preferably, the peptide nucleic acid of the present invention is derivedfrom an amino acid. More preferably, the peptide nucleic acid of thepresent invention is derived from D-lysine.

The PNAs of the present invention are synthesized by adaptation ofstandard peptide synthesis procedures, either in solution or on a solidphase.

The monomer subunits of the invention or their activated derivatives,protected by standard protecting groups, are specially designed aminoacids.

The present invention also provides a compound having formula (II):

wherein:

L is a 2,6-diaminopurine nucleobase;

R^(7′) is hydrogen or C₁-C₈ alkylamine;

E is COOH or an activated or protected derivative thereof; and

Z is NH₂ or NHPg, where Pg is an amino-protecting group.

Preferably, R^(7′) is C₃-C₆ alkylamine. More preferably, R^(7′) is C₄-C₅alkylamine. Still more preferably, R^(7′) is butyl amine.

Preferably, the carbon atom to which substituent R^(7′) is attached(identified by an asterisks) is stereochemically enriched. Preferably,the stereochemical enrichment is of R configuration.

Preferably, compound (II) of the present invention is derived from anamino acid. More preferably, compound (II) of the present invention isderived from D-lysine.

The present invention also provides a pharmaceutical compositioncomprising peptide nucleic acids of the present invention and at leastone pharmaceutically effective carrier, binder, thickener, diluent,buffer, preservative, or surface active agent.

The present invention further provides methods for enhancing thesolubility, sequence specificity and binding affinity of peptide nucleicacids by incorporating 2,6-diaminopurine nucleobases in the PNAbackbone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide examples of naturally-occurring andnon-naturally-occurring nucleobases for DNA recognition.

FIG. 2 shows the Acr¹ ligand and a PNA, Acr¹-(Taeg)₁₀-Lys-NH₂.

FIG. 3 provides a general scheme for solid phase PNA synthesisillustrating the preparation of linear unprotected PNA amides.

FIG. 4 provides a procedure for the synthesis of protected PNA synthons.

FIG. 5 provides a procedure for synthesis of thymine monomer synthonswith side chains corresponding to the common amino acids.

FIG. 6 provides a procedure for synthesis of an aminoethyl-β-alanineanalogue of thymine monomer synthon.

FIGS. 7A, 7B, and 7C are schematics showing the synthesis of PNAmonomers containing lysine.

FIG. 8 is a graph showing inhibition of HCV protein translation in an invitro translation assay.

DETAILED DESCRIPTION OF THE INVENTION

In the PNAs of the present invention having the formula (I), nucleobaseL is a naturally-occurring nucleobase attached at the position found innature, i.e., position 9 for adenine or guanine, and position 1 forthymine or cytosine, a non-naturally-occurring nucleobase (nucleobaseanalog) or a nucleobase-binding moiety, at least one of said ligands Lbeing a 2,6-diaminopurine nucleobase. Exemplary nucleobases includeadenine, guanine, thymine, cytosine, 2,6-diaminopurine, uracil,thiouracil, inosine, 5-methylcytosine, bromothymine, azaadenines andazaguanines. Some typical nucleobases and illustrative syntheticnucleobases are shown in FIGS. 1(a) and (b).

In monomer subunits according to the present invention having theformula (II), L is a naturally-occurring nucleobase or anon-naturally-occurring nucleobase which may be protected with one ormore protecting groups. Exemplary protecting groups includet-butoxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC) or2-nitrobenzyl (2Nb). Accordingly, such protecting groups may be eitheracid, base, hydrogenolytic or photolytically labile.

Preferably R^(7′) in the monomer subunit is hydrogen or C₁-C₈alkylamine.

Preferably, E in the monomer subunit is COOH or an activated derivativethereof Activation may, for example, be achieved using an acid anhydrideor an active ester derivative.

The amino acids which form the polyamide backbone may be identical ordifferent. We have found that those based on 2-aminoethylglycine areparticularly useful in the present invention.

The PNAs of the present invention may be linked to low molecular weighteffector ligands, such as ligands having nuclease activity or alkylatingactivity or reporter ligands (e.g., fluorescent, spin labels,radioactive, protein recognition ligands, for example, biotin orhaptens). PNAs may also be linked to peptides or proteins, where thepeptides have signaling activity. Exemplary proteins include enzymes,transcription factors and antibodies. The PNAs of the present inventionmay also be attached to water-soluble polymer, water-insoluble polymers,oligonucleotides or carbohydrates. When warranted, a PNA oligomer may besynthesized onto a moiety (e.g., a peptide chain, reporter, intercalatoror other type of ligand-containing group) attached to a solid support.

In monomer subunits according to the present invention having theformula (II), L is a naturally-occurring nucleobase or anon-naturally-occurring nucleobase which may be protected with one ormore protecting groups.

A compound according to the present invention having general formula(II), L is a 2,6-diaminopurine nucleobase which may be protected withone or more protecting groups. Exemplary protecting groups includet-butoxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC) or2-nitrobenzyl (2Nb). Accordingly, such protecting groups may be eitheracid, base, hydrogenolytic or photolytically labile.

Preferably R^(7′) is independently hydrogen or C₁-C₈ alkylamine.

Preferably, E in the monomer subunit is COOH or an activated derivativethereof Activation may, for example, be achieved using an acid anhydrideor an active ester derivative.

The amino acids which form the polyamide backbone may be identical ordifferent. We have found that those based on 2-aminoethylglycine areparticularly useful in the present invention.

The PNAs of the present invention may be linked to low molecular weighteffector ligands, such as ligands having nuclease activity or alkylatingactivity or reporter ligands (e.g., fluorescent, spin labels,radioactive, protein recognition ligands, for example, biotin orhaptens). PNAs may also be linked to peptides or proteins, where thepeptides have signaling activity. Exemplary proteins include enzymes,transcription factors and antibodies. The PNAs of the present inventionmay also be attached to water-soluble polymer, water-insoluble polymers,oligonucleotides or carbohydrates. When warranted, a PNA oligomer may besynthesized onto a moiety (e.g., a peptide chain, reporter, intercalatoror other type of ligand-containing group) attached to a solid support.

The PNAs of the present invention may be used for gene modulation (e.g.,gene targeted drugs), diagnostics, biotechnology and other researchpurposes. The PNAs may also be used to target RNA and single strandedDNA (ssDNA) to produce both antisense-type gene regulating moieties andas hybridization probes, e.g., for the identification and purificationof nucleic acids. Furthermore, the PNAs may be modified in such a waythat they form triple helices with double stranded DNA (dsDNA).Compounds that bind sequence-specifically to dsDNA have applications asgene targeted drugs. These compounds are extremely useful drugs fortreating diseases such as cancer, acquired immune deficiency syndrome(AIDS) and other virus infections and genetic disorders. Furthermore,these compounds may be used in research, diagnostics and for detectionand isolation of specific nucleic acids.

Gene-targeted drugs are designed with a nucleobase sequence (preferablycontaining 10-20 units) complementary to the regulatory region (thepromoter) of the target gene. Therefore, upon administration, thegene-targeted drugs bind to the promoter and prevent RNA polymerase fromaccessing the promoter. Consequently, no mRNA, and thus no gene product(protein), is produced. If the target is within a vital gene for avirus, no viable virus particles will be produced. Alternatively, thetarget region could be downstream from the promoter, causing the RNApolymerase to terminate at this position, thus forming a truncatedmRNA/protein which is nonfunctional.

Sequence-specific recognition of ssDNA by base complementaryhybridization can likewise be exploited to target specific genes andviruses. In this case, the target sequence is contained in the mRNA suchthat binding of the drug to the target hinders the action of ribosomesand, consequently, translation of the mRNA into a protein. The PNAs ofthe present invention have higher affinity for complementary ssDNA thanother currently available oligonucleotide analogs. Also PNAs of thepresent invention need not possess a net charge and can bearsubstituents that enhance aqueous solubility, which facilitates cellularuptake. In addition, the PNAs of the present invention contain amides ofnon-biological amino acids, which make them biostable and resistant toenzymatic degradation.

The PNAs of the present invention comprising C₁-C₈ alkylamine sidechains exhibit enhanced binding affinity. This is demonstrated byincreased thermal stability of the complex formed between said compoundsof the present invention and a complementary DNA strand. The PNAs of thepresent invention also exhibit enhanced solubility and sequencespecificity in binding to complementary nucleic acids.

A synthesis of PNAs according to the present invention is discussed indetail below.

Synthesis of PNA Oligomers

The principle of anchoring molecules during a reaction onto a solidmatrix is known as Solid Phase Synthesis or Merrifield Synthesis (seeMerrifield, J. Am. Chem. Soc., 1963, 85, 2149 and Science, 1986, 232,341). Established methods for the stepwise or fragment-wise solid phaseassembly of amino acids into peptides normally employ a beaded matrix ofcross-linked styrene-divinylbenzene copolymer. The cross-linkedcopolymer is formed by the pearl polymerization of styrene monomer towhich is added a mixture of divinylbenzenes. Usually, 1-2% cross-linkingis employed. Such a matrix may be used in solid phase PNA synthesis ofthe present invention (FIG. 3).

More than fifty methods for initial functionalization of the solid phasehave been described in connection with traditional solid phase peptidesynthesis (see Barany and Merrifield in “The Peptides” Vol. 2, AcademicPress, New York, 1979, pp. 1-284, and Stewart and Young, “Solid PhasePeptide Synthesis”, 2nd Ed., Pierce Chemical Company, Illinois, 1984).Reactions for the introduction of chloromethyl functionality (Merrifieldresin; via a chloromethyl methyl ether/SnCl₄ reaction), aminomethylfunctionality (via an N-hydroxymethylphthalimide reaction; Mitchell etal., Tetrahedron Lett., 1976, 3795) and benzhydrylamino functionality(Pietta et al., J. Chem. Soc., 1970, 650) are most widely used.Regardless of its nature, the purpose of introducing a functionality onthe solid phase is to form an anchoring linkage between the copolymersolid support and the C-terminus of the first amino acid to be coupledto the solid support. As will be recognized, anchoring linkages may alsobe formed between the solid support and the amino acid N-terminus. The“concentration” of a functional group present in the solid phase isgenerally expressed in millimoles per gram (mmol/g). Other reactivefunctionalities which have been initially introduced include4-methylbenzhydrylamino and 4-methoxybenzhydrylamino groups. All ofthese established methods are, in principle, useful within the contextof the present invention.

A Preferred method for PNA synthesis employs aminomethyl as the initialfunctionality. Aminomethyl is particularly advantageous as a “spacer” or“handle” group because it forms amide bonds with a carboxylic acid groupin nearly quantitative amounts. A vast number of relevant spacer- orhandle-forming bifunctional reagents have been described (see Barany etal., Int. J. Peptide Protein Res., 1987, 30, 705). Representativebifunctional reagents include 4-(haloalkyl)aryl-lower alkanoic acidssuch as 4-(bromomethyl)phenylacetic acid;BOC-aminoacyl-4-(oxymethyl)aryl-lower alkanoic acids such asBOC-aminoacyl-4-(oxymethyl)phenylacetic acid;N-BOC-p-acylbenzhydrylamines such as N-BOC-p-glutaroylbenzhydrylamine;N-BOC-4′-lower alkyl-p-acylbenzhydrylamines such asN-BOC-4′-methyl-p-glutaroylbenzhydrylamine; N-BOC-4′-loweralkoxy-p-acylbenzhydrylamines such asN-BOC-4′-methoxy-p-glutaroyl-benzhydrylamine; and4-hydroxymethylphenoxyacetic acid. One type of spacer group particularlyrelevant within the context of the present invention is thephenylacetamidomethyl (PAM) handle (Mitchell and Merrifield, J. Org.Chem., 1976, 41, 2015) which, deriving from the electron withdrawingeffect of the 4-phenylacetamidomethyl group, is about 100 times morestable than a benzyl ester linkage towards the BOC-amino deprotectionreagent trifluoroacetic acid (TFA).

Certain functionalities (e.g., benzhydrylamino, 4-methylbenzhydrylaminoand 4-methoxybenzhydrylamino), which may be incorporated for the purposeof cleavage of a synthesized PNA chain from the solid support such thatthe C-terminal of the PNA chain is released as an amide, require nointroduction of a spacer group. Any such functionality mayadvantageously be employed in the context of the present invention.

An alternative strategy concerning the introduction of spacer or handlegroups is the so-called “preformed handle” strategy (see Tam et al.,Synthesis, 1979, 955-957), which offers complete control over couplingof the first amino acid and excludes the possibility of complicationsarising from the presence of undesired functional groups not related tothe peptide or PNA synthesis. In this strategy, spacer or handle groups,of the same type as described above, are reacted with the first aminoacid desired to be bound to the solid support, the amino acid beingN-protected and optionally protected at the other side chains which arenot relevant with respect to the growth of the desired PNA chain. Thus,in those cases in which a spacer or handle group is desirable, the firstamino acid to be coupled to the solid support can either be coupled tothe free reactive end of a spacer group which has been bound to theinitially introduced functionality (for example, an aminomethyl group)or can be reacted with the spacer-forming reagent. The space-formingreagent is then reacted with the initially introduced functionality.Other useful anchoring schemes include the “multidetachable” resins (seeTam et al., Tetrahedron Lett., 1979, 4935 and J. Am. Chem. Soc., 1980,102, 611; Tam, J. Org. Chem., 1985, 50, 5291), which provide more thanone mode of release and thereby allow more flexibility in syntheticdesign.

Exemplary N-protecting groups are tert-butyloxycarbonyl (BOC) (Carpino,J. Am. Chem. Soc., 1957, 79, 4427; McKay, et al., J. Am. Chem. Soc.,1957, 79, 4686; Anderson et al., J. Am. Chem. Soc., 1957, 79, 6180) andthe 9-fluorenylmethyloxycarbonyl (FMOC) (Carpino et al., J. Am. Chem.Soc., 1970, 92, 5748 and J. Org. Chem., 1972, 37, 3404), Adoc (Hass etal., J. Am. Chem. Soc., 1966, 88, 1988), Bpoc (Sieber Helv. Chem. Acta.,1968, 51, 614), Mcb (Brady et al., J. Org. Chem., 1977, 42, 143), Bic(Kemp et al., Tetrahedron, 1975, 4624), o-nitrophenylsulfenyl (Nps)(Zervas et al., J. Am. Chem. Soc., 1963, 85, 3660) and dithiasuccinoyl(Dts) (Barany et al., J. Am. Chem. Soc., 1977, 99, 7363) as well asother groups which are known to those skilled in the art. Theseamino-protecting groups, particularly those based on the widely-usedurethane functionality, prohibit racemization (mediated bytautomerization of the readily formed oxazolinone (aziactone)intermediates (Goodman et al., J. Am. Chem. Soc., 1964, 86, 2918))during the coupling of most α-amino acids.

In addition to such amino-protecting groups, nonurethane-type ofamino-protecting groups are also applicable when assembling PNAmolecules. Thus, not only the above-mentioned amino-protecting groups(or those derived from any of these groups) are useful within thecontext of the present invention, but so are virtually anyamino-protecting groups which largely fulfill the followingrequirements: (1) stable to mild acids (not significantly attacked bycarboxyl groups); (2) stable to mild bases or nucleophiles (notsignificantly attacked by the amino group in question); (3) resistant toacylation (not significantly attacked by activated amino acids); (4) canbe substantially removed without any serious side reaction; and (5)preserves the optical integrity, if any, of the incoming amino acid uponcoupling.

The choice of side chain protecting groups, in general, depends on thechoice of the amino-protecting group, because the side chain protectinggroup must withstand the conditions of the repeated amino deprotectioncycles. This is true whether the overall strategy for chemicallyassembling PNA molecules relies on, for example, different acidstability of amino and side chain protecting groups (such as is the casefor the above-mentioned “BOC-benzyl” approach) or employs an orthogonal,that is, chemoselective, protection scheme (such as is the case for theabove-mentioned “FMOC-t-Bu” approach).

Following coupling of the first amino acid, the next stage of solidphase synthesis is the systematic elaboration of the desired PNA chain.This elaboration involves repeated deprotection/coupling cycles. Atemporary protecting group, such as BOC or FMOC, on the last coupledamino acid is quantitatively removed by a suitable treatment, forexample, by acidolysis, such as with trifluoroacetic acid in the case ofBOC, or by base treatment, such as with piperidine in the case of FMOC,so as to liberate the N-terminal amine function.

The next desired N-protected amino acid is then coupled to theN-terminal of the last coupled amino acid. This coupling of theC-terminal of an amino acid with the N-terminal of the last coupledamino acid can be achieved in several ways. For example, it can beachieved by providing the incoming amino acid in a form with thecarboxyl group activated by any of several methods, including theinitial formation of an active ester derivative such as a2,4,5-trichlorophenyl ester (Pless et al., Helv. Chim. Acta, 1963, 46,1609), a phthalimido ester (Nefkens et al., J. Am. Chem. Soc., 1961, 83,1263), a pentachlorophenyl ester (Kupryszewski, Rocz. Chem., 1961, 35,595), a pentafluorophenyl ester (Kovacs et al., J. Am. Chem. Soc., 1963,85, 183), an o-nitrophenyl ester (Bodanzsky, Nature, 1955, 175, 685), animidazole ester (Li et al., J. Am. Chem. Soc., 1970, 92, 7608), and a3-hydroxy-4-oxo-3,4-dihydroquinazoline (Dhbt-OH) ester (Konig et al.,Chem. Ber., 1973, 103, 2024 and 2034), or the initial formation of ananhydride such as a symmetrical anhydride (Wieland et al., Angew. Chem.,Int. Ed. Engl., 1971, 10, 336). Alternatively, the carboxyl group of theincoming amino acid can be reacted directly with the N-terminal of thelast coupled amino acid with the assistance of a condensation reagentsuch as, for example, dicyclohexylcarbodiimide (Sheehan et al., J. Am.Chem. Soc., 1955, 77, 1067) or derivatives thereof BenzotriazolylN-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP), “Castro'sreagent” (see Rivaille et al., Tetrahedron, 1980, 36, 3413), isrecommended when assembling PNA molecules containing secondary aminogroups. Finally, activated PNA monomers analogous to therecently-reported amino acid fluorides (Carpino, J. Am. Chem. Soc.,1990, 112, 9651) hold considerable promise to be used in PNA synthesisas well.

Following the assembly of the desired PNA chain, including protectinggroups, the next step will normally be deprotection of the amino acidmoieties of the PNA chain and cleavage of the synthesized PNA from thesolid support. These processes can take place substantiallysimultaneously, thereby providing the free PNA molecule in the desiredform. Alternatively, in cases in which condensation of two separatelysynthesized PNA chains is to be carried out, it is possible, by choosinga suitable spacer group at the start of the synthesis, to cleave thedesired PNA chains from their respective solid supports (both peptidechains still incorporating their side chain-protecting groups) andfinally removing the side chain-protecting groups after, for example,coupling the two side chain-protected peptide chains to form a longerPNA chain.

In the above-mentioned “BOC-benzyl” protection scheme, the finaldeprotection of side chains and release of the PNA molecule from thesolid support is most often carried out by the use of strong acids suchas anhydrous HF (Sakakibara et al., Bull. Chem. Soc. Jpn., 1965, 38,4921), boron tris (trifluoroacetate) (Pless et al., Helv. Chim. Acta,1973, 46, 1609) and sulfonic acids, such as trifluoromethanesulfonicacid and methanesulfonic acid (Yajima et al., J. Chem. Soc., Chem.Comm., 1974, 107). A strong acid (e.g., anhydrous HF) deprotectionmethod may produce very reactive carbocations that may lead toalkylation and acylation of sensitive residues in the PNA chain. Suchside reactions are only partly avoided by the presence of scavengerssuch as anisole, phenol, dimethyl sulfide, and mercaptoethanol. Thus,the sulfide-assisted acidolytic S_(N)2 deprotection method (Tam et al.,J. Am. Chem. Soc., 1983, 105, 6442 and J. Am. Chem. Soc., 1986, 108,5242), the so-called “low” method, which removes the precursors ofharmful carbocations to form inert sulfonium salts, is frequentlyemployed in peptide and PNA synthesis. Other methods for deprotectionand/or final cleavage of the PNA-solid support bond may includebase-catalyzed alcoholysis (Barton et al., J. Am. Chem. Soc., 1973, 95,4501), ammonolysis, hydrazinolysis (Bodanszky et al., Chem. Ind., 19641423), hydrogenolysis (Jones, Tetrahedron Lett. 1977 2853 and Schlatteret al., Tetrahedron Lett. 1977 2861)) and photolysis (Rich and Gurwara,J. Am. Chem. Soc., 1975 97, 1575)).

Finally, in contrast with the chemical synthesis of conventionalpeptides, stepwise chain building of achiral PNAs such as those based onaminoethylglycyl backbone units can start either from the N-terminus orthe C-terminus. Those skilled in the art will recognize that synthesiscommencing at the C-terminus typically employ protected amine groups andfree or activated acid groups, and syntheses commencing at theN-terminus typically employ protected acid groups and free or activatedamine groups.

Based on the recognition that most operations are identical in thesynthetic cycles of solid phase peptide synthesis (as is also the casefor solid phase PNA synthesis), a new matrix, PEPS, was recentlyintroduced (Berg et al., J. Am. Chem. Soc., 1989, 111, 8024 andInternational Patent Application WO 90/02749) to facilitate thepreparation of a large number of peptides. This matrix is comprised of apolyethylene (PE) film with pendant long-chain polystyrene (PS) grafts(molecular weight on the order of 10⁶ Daltons). The loading capacity ofthe film is as high as that of a beaded matrix, but PEPS has theadditional flexibility to suit multiple syntheses simultaneously. Thus,in a new configuration for solid phase peptide synthesis, the PEPS filmis fashioned in the form of discrete, labeled sheets, each serving as anindividual compartment. During all the identical steps of the syntheticcycles, the sheets are kept together in a single reaction vessel topermit concurrent preparation of a multitude of peptides at a rate closeto that of a single peptide synthesis by conventional methods. It isbelieved that the PEPS film support, comprising linker or spacer groupsadapted to the particular chemistry will be particularly valuable in thesynthesis of multiple PNA molecules. The synthesis of PNAs areconceptually simple because only four different reaction compartmentsare normally required, one for each of the four “pseudo-nucleotide”units. The PEPS film support has been successfully tested in a number ofPNA syntheses carried out in a parallel and substantially simultaneousfashion. The yield and quality of the products obtained from PEPS arecomparable to those obtained by using the traditional polystyrene beadsupport. Also, experiments with other geometries of the PEPS polymer,for example, non-woven felt, knitted net, sticks and microwellplates,have not indicated any limitations of the synthetic efficacy.

Two other methods for the simultaneous synthesis of large numbers ofpeptides also apply to the preparation of multiple, different PNAmolecules. The first of these methods (Geysen et al., Proc. Natl. Acad.Sci. USA, 1984, 81, 3998) utilizes acrylic acid-graftedpolyethylene-rods and 96-microtiter wells to immobilize the growingpeptide chains and to perform the compartmentalized synthesis. Whileeffective, this method is only applicable on a microgram scale. Thesecond method (Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131)utilizes a “tea bag” containing traditionally-used polymer beads. Othermethods for multiple peptide or PNA synthesis in the context of thepresent invention include the simultaneous use of two different supportswith different densities (Tregear in “Chemistry and Biology ofPeptides”, J. Meienhofer, Ed., Ann Arbor Sci. Publ., Ann Arbor, 1972,pp. 175-178), combining reaction vessels via a manifold (Gorman, Anal.Biochem., 1984, 136, 397), multicolumn solid phase synthesis (Krchnak etal., Int. J. Peptide Protein Res., 1989, 33, 209, and Holm and Meldal in“Proceedings of the 20th European Peptide Symposium”, G. Jung and E.Bayer, Eds., Walter de Gruyter & Co., Berlin, 1989, pp. 208-210) and theuse of cellulose paper (Eichler et al., Collect. Czech. Chem. Commun.,1989, 54, 1746).

Conventional cross-linked styrene/divinylbenzene copolymer matrix andthe PEPS support are preferred in the context of solid phase PNAsynthesis. Other exemplary solid supports include (1) particles basedupon copolymers of dimethylacrylamide cross-linked withN,N′-bisacryloylethylenediamine, (2) solid supports based onsilica-containing particles such as porous glass beads and silica gel,(3) composites that contain two major ingredients: a resin and anothermaterial that is also substantially inert to the reaction conditionsemployed (see Scott et al., J. Chrom. Sci., 1971, 9, 577; Kent andMerrifield, Israel J. Chem., 1978, 17, 243; and van Rietschoten in“Peptides 1974”, Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp.113-116) and (4) contiguous solid supports other than PEPS, such ascotton sheets (Lebl and Eichler, Peptide Res., 1989, 2, 232) andhydroxypropylacrylate-coated polypropylene membranes (Daniels et al.,Tetrahedron Lett., 1989, 4345).

Whether manually or automatically operated, solid phase PNA synthesis,in the context of the present invention, is normally performedbatchwise. However, most of the syntheses may be carried out equallywell in the continuous-flow mode, where the support is packed intocolumns (Bayer et al., Tetrahedron Lett., 1970, 4503; and Scott et al.,J. Chromatogr. Sci., 1971, 9, 577). With respect to continuous-flowsolid phase synthesis, the rigid poly(dimethylacrylamide)-Kieselguhrsupport (Atherton et al., J. Chem. Soc. Chem. Commun., 1981, 1151)appears to be particularly useful. Another useful configuration is theone worked out for the standard copoly(styrene-1%-divinylbenzene)support (Krchnak et al., Tetrahedron Lett., 1987, 4469).

While the solid phase technique is preferred in the present invention,other methodologies or combinations thereof may also be used. Exemplarymethodologies include (1) the classical solution phase methods forpeptide synthesis (Bodanszky, “Principles of Peptide Synthesis”,Springer-Verlag, Berlin-New York, 1984), either by stepwise assembly orby segment/fragment condensation, (2) the “liquid phase” strategy, whichutilizes soluble polymeric supports such as linear polystyrene(Shemyakin et al., Tetrahedron Lett., 1965, 2323) and polyethyleneglycol (PEG) (Mutter and Bayer, Angew. Chem., Int. Ed. Engl., 1974, 13,88), (3) random polymerization (Odian, “Principles of Polymerization”,McGraw-Hill, New York, 1970) yielding mixtures of many molecular weights(“polydisperse”) peptide or PNA molecules and (4) a technique based onthe use of polymer-supported amino acid active esters (Fridkin et al.,J. Am. Chem. Soc., 1965, 87, 4646), sometimes referred to as “inverseMerrifield synthesis” or “polymeric reagent synthesis”. In addition, itis envisaged that PNA molecules may be assembled enzymatically byenzymes such as proteases or derivatives thereof with novelspecificities (obtained, for example, by artificial means such asprotein engineering). Also, one can envision the development of “PNAligases” for the condensation of a number of PNA fragments into verylarge PNA molecules. Also, since antibodies can be generated tovirtually any molecule of interest, the recently developed catalyticantibodies (abzymes), discovered simultaneously by Tramontano et al.,Science, 1986, 234, 1566 and Pollack et al., Science, 1986, 234, 1570,should also be considered as potential candidates for assembling PNAmolecules. Thus, there has been considerable success in producingabzymes catalyzing acyl-transfer reactions (see Shokat et al., Nature,1989, 338, 269, and references therein). Finally, completely artificialenzymes, very recently pioneered by Hahn et al. (Science, 1990, 248,1544), may be developed for PNA synthesis. The design of generallyapplicable enzymes, ligases, and catalytic antibodies, capable ofmediating specific coupling reactions, should be more readily achievedfor PNA synthesis than for “normal” peptide synthesis since PNAmolecules will often be comprised of only four different amino acids(one for each of the four native nucleobases).

Likely therapeutic and prophylactic targets include herpes simplex virus(HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV),candida albicans, influenza virus, cytomegalovirus (CMV), intercellularadhesion molecules (ICAM), 5-lipoxygenase (5-LO), phospholipase A₂(PLA₂), protein kinase C (PKC), and the ras oncogene. Potentialtreatment of such targeting include ocular, labial, genital, andsystemic herpes simplex I and II infections; genital warts; cervicalcancer; common warts; Kaposi's sarcoma; AIDS; skin and systemic fungalinfections; flu; pneumonia; retinitis and pneumonitis inimmunosuppressed patients; mononucleosis; ocular, skin and systemicinflammation; cardiovascular disease; cancer; asthma; psoriasis;cardiovascular collapse; cardiac infarction; gastrointestinal disease;kidney disease; rheumatoid arthritis; osteoarthritis; acutepancreatitis; septic shock; and Crohn's disease.

In general, for therapeutic or prophylactic treatment, a patientsuspected of requiring such therapy is administered a compound of thepresent invention, commonly in a pharmaceutically acceptable carrier, inamounts and for periods of time which will vary depending upon thenature of the particular disease, it's severity and the patient'soverall condition. The peptide nucleic acids of this invention can beformulated in a pharmaceutical composition, which may include carriers,thickeners, diluents, buffers, preservatives, surface active agents andthe like. Pharmaceutical compositions may also include one or moreactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics and the like, in addition to the peptide nucleicacids.

The pharmaceutical composition may be administered in a number of waysdepending upon whether local or systemic treatment is desired, and uponthe area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal, transdermal), oral orparenteral, for example, by intravenous drip, subcutaneous,intraperitoneal or intramuscular injection or intrathecal orintraventricular administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be added.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with thecourse of treatment lasting from several days to several months or untila cure is effected or a diminution of disease state is achieved. Personsof ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates.

The present invention also pertains to the advantageous use of PNAmolecules in solid phase biochemistry (see “Solid PhaseBiochemistry—Analytical and Synthetic Aspects”, W. H. Scouten, Ed., JohnWiley & Sons, New York, 1983), notably solid phase biosystems,especially bioassays or solid phase techniques for diagnosticdetection/quantitation or affinity purification of complementary nucleicacids (see “Affinity Chromatography—A Practical Approach”, P. D. G.Dean, W. S. Johnson and F. A. Middle, Eds., IRL Press Ltd., Oxford,1986; “Nucleic Acid Hybridization—A Practical Approach”, B. D. Harnesand S. J. Higgins, IRL Press Ltd., Oxford, 1987). Current methods forperforming such bioassays or purification techniques almost exclusivelyutilize “normal” or slightly modified oligonucleotides either physicallyadsorbed or bound through a substantially permanent covalent anchoringlinkage to beaded solid supports such as cellulose, glass beads,including those with controlled porosity (Mzutani et al., J.Chromatogr., 1986, 356, 202), “Sephadex”, “Sepharose”, agarose,polyacrylamide, porous particulate alumina, hydroxyalkyl methacrylategels, diol-bonded silica, porous ceramics, or contiguous materials suchas filter discs of nylon and nitrocellulose.

All the above-mentioned methods are applicable within the context of thepresent invention. However, when possible, covalent linkage method isare preferred over the physical adsorption method, because the latterapproach may result in some of the immobilized molecules being washedout (desorbed) during the hybridization or affinity process. Theseverity of this problem will, of course, depend to a large extent onthe rate at which equilibrium between adsorbed and “free” species isestablished. In certain cases it may be virtually impossible to performa quantitative assay with acceptable accuracy and/or reproducibility.The amount of loss of adsorbed species during the treatment of thesupport with body fluids, aqueous reagents or washing media will, ingeneral, be expected to be most pronounced for species of relatively lowmolecular weight.

In contrast with oligonucleotides, PNA molecules are easier to attachonto solid supports because they contain strong nucleophilic and/orelectrophilic centers. In addition, a direct assembly ofoligonucleotides onto solid supports suffers from an extremely lowloading of the immobilized molecule (Beaucage and Caruthers, TetrahedronLett., 1981, 22, 1859; and Caruthers, Science, 1985, 232, 281). Inaddition, because it uses the alternative phosphite triester method(Letsinger and Mahadevan, J. Am. Chem. Soc., 1976, 98, 3655), which issuited for solid supports with a high surface/loading capacity, onlyrelatively short oligonucleotides can be obtained.

As for conventional solid phase peptide synthesis, however, the lattersupports are excellent materials for building up immobilized PNAmolecules. It allows the side chain-protecting groups to be removed fromthe synthesized PNA chain without cleaving the anchoring linkage holdingthe chain to the solid support. They also can be loaded onto solidsupports in large amounts, thus further increasing the capacity of thesolid phase technique.

Furthermore, certain types of studies concerning the use of PNA in solidphase biochemistry can be conducted, facilitated, or greatly acceleratedby use of the recently-reported “light-directed, spatially addressable,parallel chemical synthesis” technology (Fodor et al., Science, 1991,251, 767), a technique that combines solid phase chemistry andphotolithography to produce thousands of highly diverse, butidentifiable, permanently immobilized compounds (such as peptides) in asubstantially simultaneous way.

Synthesis of Monomer Subunits

The monomer subunits preferably are synthesized by the general schemeoutlined in FIG. 4. This involves preparation of either the methyl orethyl ester of (BOC-aminoethyl)glycine, by a protection/deprotectionprocedure as described in Examples 20-22. The synthesis of thyminemonomer is described in Examples 23-24, and the synthesis of protectedcytosine monomer is described in Example 25.

The synthesis of a protected adenine monomer involves alkylation ofadenine with ethyl bromoacetate (Example 26) and verification of theposition of substitution (i.e. position 9) by X-ray crystallography. TheN⁶-amino group is then protected with the benzyloxy-carbonyl group bythe use of the reagent N-ethyl-benzyloxycarbonylimnidazoletetrafluoroborate (Example 27). Simple hydrolysis of the product ester(Example 28) gave N⁶-benzyloxycarbonyl-9-carboxymethyl adenine, whichwas used in the standard procedure (Examples 29-30). The adenine monomerhas been built into two different PNA oligomers (Examples 52, 53, 56 and57).

For the synthesis of the protected G-monomer, the starting material,2-amino-6-chloropurine, was alkylated with bromoacetic acid (Example 31)and the chlorine atom was then substituted with a benzyloxy group(Example 32). The resulting acid was coupled to the(BOC-aminoethyl)glycine methyl ester (from Example 22) with agentPyBrop™, and the resulting ester was hydrolysed (Example 33). TheO⁶-benzyl group was removed in the final HF-cleavage step in thesynthesis of the PNA-oligomer. Cleavage was verified by mass spectrum ofthe final PNA oligomer, upon incorporation into a PNA oligomer usingdilsopropyl carbodiimide as the condensation agent (Examples 51 and 56).

Additional objects, advantages, and novel features of the presentinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are not intended tobe limiting.

General Remarks

The following abbreviations are used in the experimental examples: DMF,N,N-dimethylformamide; Tyr, tyrosine; Lys, lysine; DCC,N,N-dicyclohexyl-carbodiimide; DCU, N,N-dicyclohexylurea; THF,tetrahydrofuran; aeg, N-acetyl(2′-aminoethyl)glycine; Pfp,pentafluorophenyl; BOC, tert-butoxycarbonyl; Z, benzyloxycarbonyl; NMR,nuclear magnetic resonance; s, singlet; d, doublet; dd, doublet ofdoublets; t; triplet; q, quartet; m, multiplet; b, broad; δ, chemicalshift; ppm, parts per million (chemical shift).

NMR spectra were recorded on JEOL FX 90Q spectrometer or a Bruker 250MHz with tetramethylsilane as an internal standard. Mass spectrometrywas performed on a MassLab VG 12-250 quadropole instrument fitted with aVG FAB source and probe. Melting points were recorded on a Buchi meltingpoint apparatus and are uncorrected. N,N-Dimethylformamide was driedover 4 Å molecular sieves, distilled and stored over 4 Å molecularsieves. Pyridine (HPLC quality) was dried and stored over 4 Å molecularsieves. Other solvents used were either the highest quality obtainableor were distilled prior to use. Dioxane was passed through basic aluminaprior to use. BOC-anhydride, 4-nitrophenol, methyl bromoacetate,benzyloxycarbonyl chloride, pentafluorophenol were all obtained fromAldrich Chemical Company. Thymine, cytosine, adenine were all obtainedfrom Sigma.

Thin layer chromatography (tlc) was performed using the followingsolvent systems: (1) chloroform:triethyl amine:methanol, 7:1:2; (2)methylene chloride:methanol, 9:1; (3) chloroform:methanol:acetic acid85:10:5. Spots were visualized by UV (254 nm) and/or spraying with aninhydrin solution (3 g ninhydrin in 1000 mL of 1-butanol and 30 mL ofacetic acid), after heating at 120° C. for 5 minutes and, afterspraying, heating again.

EXAMPLE 1 Synthesis of tert-Butyl-4-nitrophenyl carbonate

Sodium carbonate (29.14 g, 0.275 mol) and 4-nitrophenol (12.75 g, 91.6mmol) were mixed with dioxane (250 mL). BOC-anhydride (2 g, 91.6 mmol)was transferred to the mixture with dioxane (50 mL). The mixture wasrefluxed for 1 h, cooled to 0° C., filtered and concentrated to a thirdof the volume, and then poured into water (350 mL) at 0° C. Afterstirring for 0.5 h, the product was collected by filtration, washed withwater, and then dried over sicapent, in vacuo. Yield 21.3 g (97%). M.p.73.0-74.5° C. (lit. 78.5-79.5° C.). Anal. for C₁₁H₁₃NO₅ found(calc.) C,55.20(55.23); H, 5.61(5.48); N, 5.82(5.85).

EXAMPLE 2 Synthesis of (N′-BOC-2′-aminoethyl)glycine (2)

The title compound was prepared by a modification of the procedure byHeimer et al. (Int. J. Pept., 1984, 23, 203-211).N-(2-Aminoethyl)glycine (1, 3 g, 25.4 mmol) was dissolved in water (50mL), dioxane (50 mL) was added, and the pH was adjusted to 11.2 with 2 Nsodium hydroxide. tert-Butyl-4-nitrophenyl carbonate (7.29 g, 30.5 mmol)was dissolved in dioxane (40 mL) and added dropwise over a period of 2h, during which time the pH was maintained at 11.2 with 2 N sodiumhydroxide. The pH was adjusted periodically to 11.2 for three more hoursand then the solution was allowed to stand overnight. The solution wascooled to 0° C. and the pH was carefully adjusted to 3.5 with 0.5 Mhydrochloric acid. The aqueous solution was washed with chloroform(3×200 mL), the pH adjusted to 9.5 with 2N sodium hydroxide and thesolution was evaporated to dryness, in vacuo (14 mm Hg). The residue wasextracted with DMF (25+2×10 mL) and the extracts filtered to removeexcess salt. This results in a solution of the title compound in about60% yield and greater than 95% purity by tlc (system 1 and visualisedwith ninhydrin, R_(f)=0.3). The solution was used in the followingpreparations of BOC-aeg derivates without further purification.

EXAMPLE 3 Synthesis of N-1-Carboxymethylthymine (4)

This procedure is different from the literature synthesis, but iseasier, gives higher yields, and leaves no unreacted thymine in theproduct. To a suspension of thymine (3, 40 g, 0.317 mol) and potassiumcarbonate (87.7 g, 0.634 mmol) in DMF (900 mL) was added methylbromoacetate (30 mL, 0.317 mmol). The mixture was stirred vigorouslyovernight under nitrogen. The mixture was filtered and evaporated todryness, in vacuo. The solid residue was treated with water (300 mL) and4 N hydrochloric acid (12 mL), stirred for 15 minutes at 0° C.,filtered, and washed with water (2×75 mL). The precipitate was treatedwith water (120 mL) and 2 N sodium hydroxide (60 mL), and was refluxedfor 10 minutes. The mixture was cooled to 0° C., filtered, and titlecompound was precipitated by the addition of 4 N hydrochloric acid (70mL). The yield after drying, in vacuo over sicapent was 37.1 g (64%).¹H-NMR: (90 MHz; DMSO-d₆): 11.33 ppm (s, 1H, NH); 7.49 (d, J=0.92 Hz,1H, ArH); 4.38 (s, 2H, CH ₂); 1.76 (d, J=0.92 Hz, T-CH ₃).

EXAMPLE 4 Synthesis of N-1-Carboxymethylthymine pentafluorophenyl ester(5)

N-1-Carboxymethylthymine (4, 10 g, 54.3 mmol) and pentafluorophenol (10g, 54.3 mmol) were dissolved in DMF (100 mL) and cooled to 5° C. in icewater. DCC (13.45 g, 65.2 mmol) was added. When the temperaturedecreased below 5° C., the ice bath was removed and the mixture wasstirred for 3 h at ambient temperature. The precipitated DCU was removedby filtration and washed twice with DMF (2×10 mL). The combined filtratewas poured into ether (1400 mL) and cooled to 0° C. Petroleum ether(1400 mL) was added and the mixture was left overnight. The titlecompound was isolated by filtration and washed thoroughly with petroleumether. Yield: 14.8 g (78%). The product was pure enough to carry out thenext reaction, but an analytical sample was obtained byrecrystallization from 2-propanol. M.p. 200.5-206° C. Anal. forC₁₃H₇F₅N₂O₄. Found(calc.) C, 44.79(44.59); H, 2.14(2.01); N, 8.13(8.00).FAB-MS: 443 (M+1+glycerol), 351 (M+1). ¹H-NMR (90 MHz; DMSO-d₆): 11.52ppm (s, 1H, NH); 7.64 (s, 1H, ArH); 4.99 (s, 2H, CH ₂); 1.76 (s, 3H, CH₃).

EXAMPLE 5 Synthesis of 1-(BOC-aeg)thymine (6)

To a DMF solution of product of Example 2 was added triethyl amine (7.08mL, 50.8 mmol) followed by N-1-carboxymethylthymine pentafluorophenylester (5, 4.45 g, 12.7 mmol). The resultant solution was stirred for 1h. The solution was cooled to 0° C. and treated with cation exchangematerial (“Dowex 50W X-8”, 40 g) for 20 minutes. The cation exchangematerial was removed by filtration, washed with dichloromethane (2×15mL), and dichloromethane (150 mL) was added. The resulting solution waswashed with saturated sodium chloride, dried over magnesium sulfate, andevaporated to dryness, in vacuo, first by a water aspirator and then byan oil pump. The residue was shaken with water (50 mL) and evaporated todryness. This procedure was repeated again. The residue then wasdissolved in methanol (75 mL) and poured into ether (600 mL) andpetroleum ether (1400 mL). After stirring overnight, the white solid wasisolated by filtration and was washed with petroleum ether. Drying oversicapent, in vacuo, gave 3.50 g (71.7%) of the title compound. M.p.142-147° C. Anal. for C₁₆H₂₄N₄O₇. Found(calc.) C, 49.59(50.00); H,6.34(6.29); N, 14.58(14.58). ¹H-NMR (250 MHz, DMSO-d₆): Due to thelimited rotation around the secondary amide bond several of the signalswere doubled in the ratio 2:1 (indicated in the list by mj. for majorand mi. for minor): 12.73 ppm (b, 1H, CO₂H); 11.27 ppm (s, mj., imide);11.25 ppm (s, mi., imide); 7.30 ppm (s, mj., ArH); 7.26 ppm (s, mi.,ArH); 6.92 ppm (unres. t, mj., BOC-NH); 6.73 ppm (unres. t; mi.,BOC-NH); 4.64 ppm (s, mj., T-CH₂—CO—); 4.47 ppm (s, mi., T-CH₂—CO—);4.19 ppm (s, mi., CONRCH ₂CO₂H); 3.97 ppm (s, mj., CONRCH ₂CO₂H);3.41-2.89 ppm (unres. m, —CH₂CH₂— and water); 1.75 ppm (s,3H, T-CH₃);1.38 ppm (s, 9H, t-Bu). ¹³C-NMR: 170.68 ppm (CO); 170.34 (CO); 167.47(CO); 167.08 (CO); 164.29 (CO); 150.9 (C5″); 141.92 (C6″); 108.04 (C2′);77.95 and 77.68 (Thy-CH ₂CO); 48.96, 47.45 and 46.70 (—CH₂ CH₂— and NCH₂CO₂H); 37.98 (Thy-CH ₃); 28.07 (t-Bu). FAB-MS: 407 (M+Na⁺); 385 (M+H⁺).

EXAMPLE 6 Synthesis of 1-(BOC-aeg)thymine pentafluorophenyl ester (7,BOC-Taeg.OPfp)

1-(BOC-aeg)thymine (6) (2 g, 5.20 mmol) was dissolved in DMF (5 mL) andmethylene chloride (15 mL) was added. Pentafluorophenol (1.05 g, 5.72mmol) was added and the solution was cooled to 0° C. in an ice bath. DDCthen was added (1.29 g, 6.24 mmol) and the ice bath was removed after 2minutes. After 3 h of stirring at ambient temperature, the precipitatedDCU was removed by filtration and washed with methylene chloride. Thecombined filtrate was washed twice with aqueous sodium hydrogencarbonate and once with saturated sodium chloride, dried over magnesiumsulfate, and evaporated to dryness, in vacuo. The solid residue wasdissolved in dioxane (150 mL) and poured into water (200 mL) at 0° C.The title compound was isolated by filtration, washed with water, anddried over sicapent, in vacuo. Yield: 2.20 g (77%). An analytical samplewas obtained by recrystallisation from 2-propanol. M.p. 174-175.5° C.Analysis for C₂₂H₂₃N₄O₇F₅, found(calc.): C, 48.22(48.01); H, 4.64(4.21);N, 9.67(10.18). ¹H-NMR (250 MHz, CDCl₃): Due to the limited rotationaround the secondary amide bond several of the signals were doubled inthe ratio 6:1 (indicated in the list by mj. for major and mi. forminor): 7.01 ppm (s, mi., ArH); 6.99 ppm (s, mj., ArH); 5.27 ppm (unres.t, BOC—NH); 4.67 ppm (s, mj., T-CH₂—CO—); 4.60 ppm (s, mi., T-CH₂—CO—);4.45 ppm (s, mj., CONRCH ₂CO₂Pfp); 4.42 ppm (s, mi., CONRCH ₂CO₂Pfp);3.64 ppm (t, 2H, BOC—NHCH₂CH ₂—); 3.87 ppm (“q”, 2H, BOC—NHCH ₂CH₂—);1.44(s,9H,t-Bu). FAB-MS: 551 (10; M+1); 495 (10; M+1-tBu); 451 (80;—BOC).

EXAMPLE 7 Synthesis of N⁴-Benzyloxycarbonyl cytosine (9)

Over a period of about 1 h, benzyloxycarbonyl chloride (52 mL, 0.36 mol)was added dropwise to a suspension of cytosine (8, 20 g, 0.18 mol) indry pyridine (1000 mL) at 0° C. under nitrogen in oven-dried equipment.The solution then was stirred overnight, after which the pyridinesuspension was evaporated to dryness, in vacuo. Water (200 mL) and 4 Nhydrochloric acid were added to reach pH ˜1. The resulting whiteprecipitate was filtered off, washed with water and partially dried byair suction. The moist precipitate was refluxed with absolute ethanol(500 mL) for 10 minutes, cooled to 0° C., filtered, washed thoroughlywith ether, and dried, in vacuo. Yield 24.7 g (54%). M.p.>250° C. Anal.for C₁₂H₁₁N₃O₃. Found(calc.); C, 58.59(58.77); H, 4.55(4.52); N,17.17(17.13). No NMR spectra were recorded since it was not possible toget the product dissolved.

EXAMPLE 8 Synthesis of N⁴-Benzyloxycarbonyl-N¹-carboxymethyl cytosine(10)

In a three-necked round bottom flask equipped with mechanical stirringand nitrogen inlet was placed methyl bromacetate (7.82 mL, 82.6 mmol)and a suspension of N⁴-benzyloxycarbonyl-cytosine (9, 21 g, 82.6 mmol)and potassium carbonate (11.4 g, 82.6 mmol) in dry DMF (900 mL). Themixture was stirred vigorously overnight, filtered, and evaporated todryness, in vacuo. Water (300 mL) and 4 N hydrochloric acid (10 mL) wereadded, the mixture was stirred for 15 minutes at 0° C., filtered, andwashed with water (2×75 mL). The isolated precipitate was treated withwater (120 mL), 2N sodium hydroxide (60 mL), stirred for 30 minutes,filtered, cooled to 0° C., and 4 N hydrochloric acid (35 mL) was added.The title compound was isolated by filtration, washed thoroughly withwater, recrystallized from methanol (1000 mL) and washed thoroughly withether. This afforded 7.70 g (31%) of pure title compound. The motherliquor from recrystallization was reduced to a volume of 200 mL andcooled to 0° C. This afforded an additional 2.30 g of a material thatwas pure by tlc but had a reddish color. M.p. 266-274° C. Anal. forC₁₄H₁₃N₃O₅. Found(calc.); C, 55.41(55.45); H, 4.23(4.32); N,14.04(13.86). ¹H-NMR (90 MHz; DMSO-d₆): 8.02 ppm (d,J=7.32 Hz, 1H, H-6);7.39 (s, 5H, Ph); 7.01 (d, J=7.32 Hz, 1H, H-5); 5.19 (s, 2H, PhCH ₂—);4.52 (s, 2H).

EXAMPLE 9 Synthesis of N⁴-Benzyloxycarbonyl-N¹-carboxymethyl-cytosinepentafluorophenyl ester (11)

N⁴-Benzyloxycarbonyl-N¹-carboxymethyl-cytosine (10, 4 g, 13.2 mmol) andpentafluorophenol (2.67 g, 14.5 mmol) were mixed with DMF (70 mL),cooled to 0° C. with ice-water, and DCC (3.27 g, 15.8 mmol) was added.The ice bath was removed after 3 minutes and the mixture was stirred for3 h at room temperature. The precipitated DCU was removed by filtration,washed with DMF, and the filtrate was evaporated to dryness, in vacuo(0.2 mm Hg). The solid residue was treated with methylene chloride (250mL), stirred vigorously for 15 minutes, filtered, washed twice withdiluted sodium hydrogen carbonate and once with saturated sodiumchloride, dried over magnesium sulfate, and evaporated to dryness, invacuo. The solid residue was recrystallized from 2-propanol (150 mL) andthe crystals were washed thoroughly with ether. Yield 3.40 g (55%). M.p.241-245° C. Anal. for C₂₀H₁₂N₃F₅O₅. Found(calc.); C, 51.56(51.18); H,2.77(2.58); N, 9.24(8.95).¹H-NMR (90 MHz; CDCl₃): 7.66 ppm (d, J=7.63Hz, 1H, H-6); 7.37 (s, 5H, Ph); 7.31 (d, J=7.63 Hz, 1H, H-5); 5.21 (s,2H, PhCH ₂—); 4.97 (s, 2H, NCH ₂—). FAB-MS: 470 (M+1)

EXAMPLE 10 Synthesis of N⁴-Benzyloxycarbonyl-1-BOC-aeg-cytosine (12)

To a solution of (N—BOC-2-aminoethyl)glycine (2) in DMF, prepared asdescribed above, was added triethyl amine (7 mL, 50.8 mmol) andN⁴-benzyloxycarbonyl-N¹-carboxymethyl-cytosine pentafluorophenyl ester(11, 2.7 g, 5.75 mmol). After stirring the solution for 1 h at roomtemperature, methylene chloride (150 mL), saturated sodium chloride (250mL), and 4 N hydrochloric acid to pH ˜1 were added. The organic layerwas separated and washed twice with saturated sodium chloride, driedover magnesium sulfate, and evaporated to dryness, in vacuo, first witha water aspirator and then with an oil pump. The oily residue wastreated with water (25 mL) and was again evaporated to dryness, invacuo. This procedure then was repeated. The oily residue (2.8 g) wasthen dissolved in methylene chloride (100 mL), petroleum ether (250 mL)was added, and the mixture was stirred overnight. The title compound wasisolated by filtration and washed with petroleum ether. Tlc (system 1)indicated substantial quantities of pentafluorophenol, but no attemptwas made to remove it. Yield: 1.72 g (59%). M.p. 156° C.(decomp.).¹H-NMR (250 MHz, CDCl₃): Due to the limited rotation around thesecondary amide bond several of the signals were doubled in the ratio2:1 (indicated in the list by mj. for major and mi. for minor): 7.88 ppm(dd, 1H H-6); 7.39 (m, 5H Ph); 7.00 (dd, 1H, H-5); 6.92 (b, 1H, BOC—NH);6.74 (b, 1H, ZNH)-?; 5.19 (s, 2H, Ph-CH ₃); 4.81 ppm (s, mj.,Cyt-CH₂—CO—); 4.62 ppm (s, mi., Cyt-CH₂—CO—); 4.23 (s, mi., CONRCH₂CO₂H); 3.98 ppm (s, mj., CONRCH ₂CO₂H); 3.42-3.02 (unres. m, —CH₂CH₂—and water);1.37 (s, 9H, t-Bu). FAB-MS: 504 (M+1); 448 (M+1-t-Bu).

EXAMPLE 11 Synthesis of N⁴-Benzyloxycarbonyl-1-BOC-aeg-cytosinepentafluorophenyl ester (13)

N⁴-Benzyloxycarbonyl-1-BOC-aeg-cytosine (12, 1.5 g, 2.98 mmol) andpentafluorophenol (548 mg, 2.98 mmol) was dissolved in DMF (10 mL).Methylene chloride (10 mL) was added, the reaction mixture was cooled to0° C. in an ice bath, and DCC (676 mg, 3.28 mmol) was added. The icebath was removed after 3 minutes and the mixture was stirred for 3 h atambient temperature. The precipitate was isolated by filtration andwashed once with methylene chloride. The precipitate was dissolved inboiling dioxane (150 mL) and the solution was cooled to 15° C., wherebyDCU precipitated. The precipitated DCU was removed by filtration and theresulting filtrate was poured into water (250 mL) at 0° C. The titlecompound was isolated by filtration, was washed with water, and driedover sicapent, in vacuo. Yield 1.30 g (65%). Analysis for C₂₉H₂₈N₅O₈F₅.Found(calc.); C, 52.63(52.02); H, 4.41(4.22); N, 10.55(10.46). ¹H-NMR(250 MHz; DMSO-d₆): showed essentially the spectrum of the above acid,most probably due to hydrolysis of the ester. FAB-MS: 670 (M+1); 614(M+1-t-Bu).

EXAMPLE 12 Synthesis of 4-chlorocarboxy-9-chloroacridine

4-Carboxyacridone (6.25 g, 26.1 mmol), thionyl chloride (25 mL) and 4drops of DMF were heated gently under a flow of nitrogen until all solidmaterial had dissolved. The solution then was refluxed for 40 minutes.The solution was cooled and excess thionyl chloride was removed invacuo. The last traces of thionyl chloride were removed by coevaporationwith dry benzene (dried over Na—Pb) twice. The remaining yellow powderwas used directly in the next reaction.

EXAMPLE 13 Synthesis of4-(5-methoxycarbonylpentylamidocarbonyl)-9-chloroacridine

Methyl 6-aminohexanoate hydrochloride (4.7 g, 25.9 mmol) was dissolvedin methylene chloride (90 mL), cooled to 0° C., triethyl amine (15 mL)was added, and the resulting solution was then immediately added to theacid chloride from Example 12. The round bottom flask containing theacid chloride was cooled to 0° C. in an ice bath. The mixture wasstirred vigorously for 30 minutes at 0° C. and 3 h at room temperature.The resulting mixture was filtered to remove the remaining solids, whichwere washed with methylene chloride (20 mL). The reddish-brown methylenechloride filtrate was subsequently washed twice with saturated sodiumhydrogen carbonate, once with saturated sodium chloride, dried overmagnesium sulfate, and evaporated to dryness, in vacuo. To the resultingoily residue was added dry benzene (35 mL) and ligroin (60-80° C., driedover Na—Pb). The mixture was heated to reflux. Activated carbon andcelite were added and the mixture refluxed for 3 minutes. Afterfiltration, the title compound crystallised upon cooling with magneticstirring. It was isolated by filtration and washed with petroleum ether.The product was stored over solid potassium hydroxide. Yield 5 g (50%).

EXAMPLE 14 Synthesis of4-(5-methoxycarbonylpentyl)amidocarbonyl-9-[6′-(4″-nitrobenzamido)-hexylamino]-aminoacridine

4-(5-Methoxycarbonylpentylamidocarbonyl)-9-chloroacridine (1.3 g, 3.38mmol) and phenol (5 g) were heated to 80° C. for 30 minutes under a flowof nitrogen, after which 6-(4′-nitrobenzamido)-1-hexylamine (897 mg,3.38 mmol) was added. The temperature was then increased to 120° C. for2 h. The reaction mixture was cooled and methylene chloride (80 mL) wasadded. The resulting solution was washed three times with 2 N sodiumhydroxide (60 mL portions) and once with water, dried over magnesiumsulfate, and evaporated to dryness, in vacuo. The resulting red oil (1.8g) was dissolved in methylene chloride (40 mL) and cooled to 0° C. Ether(120 mL) was added and the resultant solution was stirred overnight.This resulted in a mixture of solid material and an oil. The solid wasisolated by filtration. The solid and the oil were re-dissolved inmethylene chloride (80 mL) and added dropwise to cold ether (150 mL).After 20 minutes of stirring, the title compound was isolated byfiltration as orange crystals. The product was washed with ether anddried in vacuo over potassium hydroxide. Yield 1.6 g (77%). M.p.145-147° C.

EXAMPLE 15 Synthesis of4-(5-carboxypentyl)amidocarbonyl-9-[6′-(4″-nitrobenzamido)-hexylamino]-aminoacridine

4-(5-Methoxycarbonylpentyl)amidocarbonyl-9-[6′-(4″-nitrobenzamido)hexylamino]aminoacridine(503 mg, 0.82 mmol) was dissolved in DMF (30 mL), and 2 N sodiumhydroxide (30 mL) was added. After stirring for 15 minutes, 2 Nhydrochloric acid (35 mL) and water (50 mL) were added at 0° C. Afterstirring for 30 minutes, the solution was decanted, leaving an oilysubstance which was dissolved in boiling methanol (150 mL), filtered andconcentrated to a third of the volume. To the methanol solution wereadded ether (125 mL) and 5-6 drops of HCl in ethanol. The solution wasdecanted after 1 h of stirring at 0° C. The oily substance wasredissolved in methanol (25 mL) and precipitated with ether (150 mL).The title compound was isolated as yellow crystals after stirringovernight. Yield: 417 mg (80%). M.p. 173° C. (decomp.).

EXAMPLE 16

(a) Synthesis of4-(5-pentafluorophenyloxycarbonylpentyl)-amidocarbonyl-9-[6′-(4″-nitrobenzamido)-hexylamino]-aminoacridine(Acr¹OPfp)

The acid from above, Example 15, (300 mg, 0.48 mmol) was dissolved inDME (2 mL) and methylene chloride (8 mL) was added. Pentafluorophenol(97 mg, 0.53 mmol), transferred with 2×2 mL of the methylene chloridesolution, was added. The resulting solution was cooled to 0° C. afterwhich DCC (124 mg, 0.6 mmol) was subsequently added. The ice bath wasremoved after 5 minutes and the mixture was stirred overnight. Theprecipitated DCU was removed by centrifugation and the centrifugate wasevaporated to dryness, in vacuo, first by a water aspirator and then byan oil pump. The residue was dissolved in methylene chloride (20 mL),filtered, and evaporated to dryness, in vacuo. The residue was againdissolved in methylene chloride and petroleum ether (150 mL). A 1 mLaliquot of 5 M HCl in ether was added. The solvent was removed bydecanting after 30 minutes of stirring at 0° C. The residual oilysubstance was dissolved in methylene chloride (100 mL). Petroleum ether(150 mL) was added and the mixture was stirred overnight. The yellowprecipitated crystalline material was isolated by filtration and washedwith copious amounts of petroleum ether. Yield (after drying): 300 mg(78%). M.p. 97.5° C. (decomp.) All samples showed satisfactory elementalanalysis, ¹H- and ¹³C-NMR and mass spectra.

b) Experimental Procedure for the Synthesis of PNAs (FIG. 3)

Materials: BOC-Lys (ClZ),benzhydrylamine-copoly(styrene-1%-divinylbenzene) resin (BHA resin), andp-methylbenzhydrylamine-copoly(styrene-1%-divinylbenzene) resin (MBHAresin) were purchased from Peninsula Laboratories. Other reagents andsolvents were: Biograde trifluoroacetic acid from Halocarbon Products;diisopropylethylamine (99%; was not further distilled) andN-acetylimidazole (98%) from Aldrich; H₂O was distilled twice; anhydrousHF from Union Carbide; synthesis grade N,N-dimethylformamide andanalytical grade methylene chloride (was not further distilled) fromMerck; HPLC grade acetonitrile from LabScan; purum grade anisole,N,N′-dicyclohexylcarbodiimide, and puriss. grade 2,2,2-trifluoroethanolfrom Fluka.

General Methods and Remarks

Except where otherwise stated, the following applies. The PNA compoundswere synthezised by the stepwise solid phase approach (Merrifield, J.Am. Chem. Soc., 1963, 85, 2149) employing conventional peptide chemistryutilizing the TFA-labile tert-butyloxycarbonyl (BOC) group for“temporary” N-protection (Merrifield, J. Am. Chem. Soc., 1964, 86, 304)and the more acid-stable benzyloxycarbonyl (Z) and2-chlorobenzyloxycarbonyl (ClZ) groups for “permanent” side chainprotection. To obtain C-terminal amides, the PNAs were assembled ontothe HF-labile BHA or MBHA resins (the MBHA resin has increasedsusceptibility to the final HF cleavage relative to the unsubstitutedBHA resin (Matsueda et al., Peptides, 1981, 2, 45). All reactions(except HF reactions) were carried out in manually operated standardsolid phase reaction vessels fitted with a coarse glass frit (Merrifeldet al., Biochemistry, 1982, 21, 5020). The quantitative ninhydrinreaction (Kaiser test), originally developed by Sarin et al. (Anal.Biochem., 1981, 117, 147) for peptides containing “normal” amino acids,was successfully appplied (see Table I-III) using the “normally”employed effective extinction coefficient ε=15000 M⁻¹cm⁻¹ for allresidues to determine the completeness of the individual couplings aswell as to measure the number of growing peptide chains. The theoreticalsubstitution S_(n−1) upon coupling of residue number n (assuming bothcomplete deprotection and coupling as well as neither chain terminationnor loss of PNA chains during the synthetic cycle) is calculated fromthe equation:

S _(n) =S _(n−1)×(1+(S _(n−1) ×ΔMW×10⁻³ mmol/mol))⁻¹

where ΔMW is the gain in molecular weight ([ΔMW]=g/mol) and S_(n−1) isthe theoretical substitution upon coupling of the preceding residue n−1([S]=mmol/g). The estimated value (%) on the extent of an individualcoupling is calculated relative to the measured substitution (unless Swas not determined) and include correction for the number of remainingfree amino groups following the previous cycle. HF reactions werecarried out in a Diaflon HF apparatus from Toho Kasei (Osaka, Japan).Vydac C₁₈ (5 μm, 0.46×25 cm and 5 μm, 1×25 cm) reverse-phase columns,respectively were used for analytical and semi-preparative HPLC on anSP8000 instrument. Buffer A was 5 vol % acetonitrile in water containing445 μl trifluoroacetic acid per liter, and buffer B was 60 vol %acetonitrile in water containing 390 μL trifluoroacetic acid per liter.The linear gradient was 0-100% of buffer B in 30 minutes, flow rateswere 1.2 mL/minute (analytical) and 5 mL/minute (semi-preparative). Theeluents were monitored at 215 nm (analytical) and 230 nm(semi-preparative). Molecular weights of the PNAs were determined by²⁵²Cf plasma desorption time-of-flight mass spectrometry from the meanof the most abundant isotopes.

EXAMPLE 17

Solid Phase Synthesis of Acr¹-[Taeg]₁₅-NH₂ and Shorter Derivatives

(a) Stepwise Assembly of BOC-[Taeg]₁₅-BHA Resin

The synthesis was initiated on 100 mg of preswollen and neutralized BHAresin (determined by the quantitative ninhydrin reaction to contain 0.57mmol NH₂/g) employing single couplings (“Synthetic Protocol 1”) using3.2 equivalents of BOC-Taeg-OPfp in about 33% DMF/CH₂Cl₂. The individualcoupling reactions were carried out by shaking for at least 12 h in amanually operated 6 mL standard solid phase reaction vessel andunreacted amino groups were blocked by acetylation at selected stages ofthe synthesis. The progress of chain elongation was monitored at severalstages by the quantitative ninhydrin reaction (see Table I). Portions ofprotected BOC-[Taeg]₅-BHA, BOC-[Taeg]₁₀-BHA, and BOC-[Taeg]₁₅-BHA resinswere taken out after assembling 5, 10, and 15 residues, respectively.

Remaining Free Amino Estimated Substitution After Groups After ExtentDeprotection (μmol/g) of Synthetic Residue (mmol/g) Single Coupling StepCoupled Measd. Theor. Coupling Acetyln. (%) “0” 0.57 1 BOC-Taeg ND 0.501.30 <99.7 2 BOC-Taeg ND 0.44 1.43 <99.9 3 BOC-Taeg 0.29 0.39 3.33 99.34 BOC-Taeg 0.27 0.35 13.30 96.3 5 BOC-Taeg 0.26 0.32 8.33 >99.9 6BOC-Taeg ND 0.30 7.78 >99.9 7 BOC-Taeg ND 0.28 13.81 7.22 <97.8 8BOC-Taeg ND 0.26 14.00 <99.9 9 BOC-Taeg ND 0.24 30.33 93.2 10  BOC-Taeg0.16 0.23 11.67 2.67 >99.9 11  BOC-Taeg ND 0.21 4.58 >99.9 12  BOC-TaegND 0.20 5.87 <99.4 13  BOC-Taeg ND 0.19 1.67 >99.9 14  BOC-Taeg ND 0.1814.02 <93.1 15  BOC-Taeg 0.07 0.17 420 3.33 >99.9 ND = Not Determined

(b) Synthesis of Acr¹-[Taeg]₁₅-BHA Resin

Following deprotection of the residual BOC-[Taeg]₁₅-BHA resin (estimateddry weight is about 30 mg, ˜0.002 mmol growing chains), theH-[Taeg]₁₅-BHA resin was reacted with about 50 equivalents (80 mg, 0.11mmol) of Acr¹-OPfp in 1 mL of about 66% DMF/CH₂Cl₂ (i.e., a 0.11 Msolution of the pentafluorophenylester) in a 3 mL solid phase reactionvessel. As judged by a qualitative ninhydrin reaction, coupling of theacridine moiety was close to quantitative.

(c) Cleavage, Purification, and Identification of H-[Taeg]₅-NH₂

A portion of protected BOC-[Taeg]₅-BHA resin was treated with 50%trifluoroacetic acid in methylene chloride to remove the N-terminal BOCgroup (which is a precursor of the potentially harmful tert-butylcation) prior to the HF cleavage. Following neutralization and washing(performed in a way similar to those of steps 2-4 in “Synthetic Protocol1”), and drying for 2 h in vacuum, the resulting 67.1 mg (dry weight) ofH-[Taeg]₅-BHA resin was cleaved with 5 mL of HF:anisole (9:1, v/v)stirring at 0° C. for 60 minutes. After removal of HF, the residue wasstirred with dry diethyl ether (4×15 mL, 15 minutes each) to removeanisole, filtered under gravity through a fritted glass funnel, anddried. The PNA was then extracted into a 60 mL (4×15 mL, stirring 15minutes each) 10% aqueous acetic acid solution. Aliquots of thissolution were analyzed by analytical reverse-phase HPLC to establish thepurity of the crude PNA. The main peak at 13 minutes accounted for about93% of the total absorbance. The remaining solution was frozen andlyophilized to afford about 22.9 mg of crude material. Finally, 19 mg ofthe crude product was purified from five batches, each containing 3.8 mgin 1 mL of H₂O. The main peak was collected by use of a semi-preparativereverse-phase column. Acetonitrile was removed on a speed vac and theresidual solution was frozen (dry ice) and subsequently lyophilized togive 13.1 mg of >99% pure H-[Taeg]₅-NH₂. The PNA molecule readilydissolved in water and had the correct molecular weight based on massspectral determination. For (M+H)⁺ the calculated m/z value was 1349.3and the measured m/z value was 1347.8.

(d) Cleavage, Purification, and Identification of H-[Taeg]₁₀-NH₂

A portion of protected BOC-[Taeg]₁₀-BHA resin was treated as describedin section (c) to yield 11 mg of crude material upon HF cleavage of 18.9mg dry H-[Taeg]₁₀-BHA resin. The main peak at 15.5 minutes accounted forabout 53% of the total absorbance. About 1 mg of the crude product waspurified repeatedly (for reasons described below) to give approximately0.1 mg of at least 80% but presumably >99% pure H-[Taeg]₁₀-NH₂. A ratherbroad tail eluting after the target peak and accounting for about 20% ofthe total absorbance could not be removed (only slightly reduced) uponthe repeated purification. Judged by the mass spectrum, which onlyconfirms the presence of the correct molecular weight H-[Taeg]₁₀-NH₂,the tail phenomonen is ascribed to more or less well-definedaggregational/conformational states of the target molecule. Therefore,the crude product is likely to contain more than the above-mentioned 53%of the target molecule. H-[Taeg]₁₀-NH₂ is readily dissolved in water.For (M+H)⁺ the calculated m/z value was 2679.6 and the measured m/zvalue was 2681.5.

(e) Cleavage, Purification, and Identification of H-[Taeg]₁₅-NH₂

A portion of protected BOC-[Taeg]₁₅-BHA resin was treated as describedin section (c) to yield 3.2 mg of crude material upon HF cleavage of13.9 mg dry H-[Taeg]₁₅-BHA resin. The main peak at 22.6 minutes waslocated in a broad bulge accounting for about 60% of the totalabsorbance (FIG. 12a). Again (see the preceding section), this bulge isascribed to aggregational/conformational states of the target moleculeH-[Taeg]₁₅-NH₂ since mass spectral analysis of the collected “bulge” didnot significantly reveal the presence of other molecules. All of thecrude product was purified collecting the “bulge” to give approximately2.8 mg material. For (M+Na)⁺ the calculated m/z value was 4033.9 and themeasured m/z value was 4032.9.

(f) Cleavage, Purification, and Identification of Acr¹-[Taeg]₁₅-NH₂

A portion of protected Acr¹-[Taeg]₁₅-BHA resin was treated as describedin section (b) to yield 14.3 mg of crude material upon HF cleavage of29.7 mg dry Acr¹-[Taeg]₁₅-BHA resin. Taken together, the main peak at23.7 minutes and a “dimer” (see below) at 29.2 minutes accounted forabout 40% of the total absorbance (FIG. 12b). The crude product waspurified repeatedly to give approximately 1 mg of presumably >99% pureAcr¹-[Taeg]₁₅-NH₂ “contaminated” with self-aggregated molecules elutingat 27.4 minutes, 29.2 minutes, and finally as a huge broad bulge elutingwith 100% buffer B (FIG. 12c). This interpretation is in agreement withthe observation that those peaks grow upon standing (for hours) inaqueous acetic acid solution, and finally precipitate outquantitatively. For (M+H)⁺ the calculated m/z value was 4593.6 and themeasured m/z value was 4588.7.

(g) Synthetic Protocol 1

(1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 3 mL, 3×1 minute and1×30 minutes; (2) washing with CH₂Cl₂, 3 mL, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 3 mL, 3×2 minutes; (4)washing with CH₂Cl₂, 3 mL, 6×1 minute, and drain for 1 minute; (5) 2-5mg sample of PNA-resin may be removed and dried thoroughly for aquantitative ninhydrin analysis to determine the substitution; (6)addition of 3.2 equiv. (0.18 mmol, 100 mg) BocTaeg-OPfp dissolved in 1mL of CH₂Cl₂ followed by addition of 0.5 mL of DMF (final concentrationof pentafluorophenylester ˜0.12 M); the coupling reaction was allowed toproceed for a total of 12-24 h shaking at room temperature; (7) washingwith DMF, 3 mL, 1×2 minutes; (8) washing with CH₂Cl₂, 3 mL, 4×1 minute;(9) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 3 mL, 2×2 minutes; (10)washing with CH₂Cl₂, 3 mL, 6×1 minutes; (11) 2-5 mg sample of protectedPNA-resin is taken out for a rapid qualitative ninhydrin test andfurther 2-5 mg is dried thoroughly for a quantitative ninhydrin analysisto determine the extent of coupling (after cycles 7, 10, and 15unreacted amino groups were blocked by acetylation with N-acetylimidazolin methylene chloride).

EXAMPLE 18

Solid Phase Synthesis of Acr¹-[Taeg]₁₅-Lys-NH₂ and Shorter Derivatives

(a) Stepwise Assembly of BOC-[Taeg]₁₅-Lys(ClZ)-BHA Resin

The synthesis was initiated by a quantitative loading (standard DCC insitu coupling in neat CH₂Cl₂) of BOC-Lys(ClZ) onto 100 mg of preswollenand neutralized BHA resin (0.57 mmol NH₂/g). Further extension of theprotected PNA chain employed single couplings (“Synthetic Protocol 2”)for cycles 1 to 5 and cycles 10 to 15 using 3.2 equivalents ofBOC-Taeg-OPfp in about 33% DMF/CH₂Cl₂. Cycles 5 to 10 employed anadditional DCC (i.e., in situ) coupling of the free acid BOC-Taeg-OH inabout 33% DMF/CH₂Cl₂. All coupling reactions were carried out by shakingfor at least 12 h in a manually operated 6 mL standard solid phasereaction vessel. Unreacted amino groups were blocked by acetylation atthe same stages of the synthesis as was done in Example 17. Portions ofprotected BOC-[Taeg]₅-Lys(ClZ)-BHA and BOC-[Taeg]₁₀-Lys(ClZ)-BHA resinswere removed after assembling 5 and 10 PNA residues, respectively. Asjudged by the analytical HPLC chromatogram of the crude cleavage productfrom the BOC-[Taeg]₁₀-Lys(ClZ)-BHA resin (see section (e)), anadditional “free acid” coupling of PNA residues 5 to 10 gave nosignificant improvement of the synthetic yield as compared to thethroughout single-coupled residues in Example 17.

(B) Synthesis of Acr¹-[Taeg]₁₀-Lys(ClZ)-BHA Resin

Following deprotection of a portion of BOC-[Taeg]₁₀-Lys(ClZ)-BHA resin(estimated dry weight is about 90 mg, ˜0.01 mmol growing chains), theH-[Taeg]₁₅-BHA resin was reacted with about 20 equivalents (141 mg, 0.19mmol) of Acr¹-OPfp in 1 mL of about 66% DMF/CH₂Cl₂ in a 3 mL solid phasereaction vessel. As judged by a qualitative ninhydrin reaction, couplingof the acridine moiety was close to quantitative.

(c) Synthesis of Acr¹-[Taeg]₁₅-Lys(ClZ)-BEA Resin

Following deprotection of the residual BOC-[Taeg]₁₅-Lys(ClZ)-BHA resin(estimated dry weight about 70 mg, ˜0.005 mmol growing chains), theH-[Taeg]₁₅-Lys(ClZ)-BHA resin was reacted with about 25 equivalents (91mg, 0. 12 mmol) of Acr¹-OPfp in 1 mL of about 66% DMF/CH₂Cl₂ in a 3 mLsolid phase reaction vessel. As judged by a qualitative ninhydrinreaction, coupling of the acridine moiety was close to quantitative.

(d) Cleavage, Purification, and Identification of H-[Taeg]₅-Lys-NH₂

A portion of protected BOC-[Taeg]₅-Lys(ClZ)-BHA resin was treated asdescribed in Example 17(c) to yield 8.9 mg of crude material upon HFcleavage of 19 mg dry H-[Taeg]₅-Lys(ClZ)-BHA resin. The main peak at12.2 minutes (eluted at 14.2 minutes if injected from an aqueoussolution instead of the 10% aqueous acetic acid solution) accounted forabout 90% of the total absorbance. About 2.2 mg of the crude product waspurified to give approximately 1.5 mg of 99% pure H-[Taeg]₅-Lys-NH₂

(e) Cleavage, Purification, and Identification of H-[Taeg]₁₀-Lys-NH₂

A portion of protected BOC-[Taeg]₁₀-Lys(ClZ)-BHA resin was treated asdescribed in Example 17(c) to yield 1.7 mg of crude material upon HFcleavage of 7 mg dry H-[Taeg]₁₀-Lys(ClZ)-BHA resin. The main peak at15.1 minutes (eluted at 17 minutes if injected from an aqueous solutioninstead of the 10% aqueous acetic acid solution) accounted for about 50%of the total absorbance. About 1.2 mg of the crude product was purifiedto give approximately 0.2 mg of >95% pure H-[Taeg]₁₀-Lys-NH₂ (FIG. 4).For (M+H)⁺ the calculated m/z value was 2807.8 and the measured m/zvalue was 2808.2.

(f) Cleavage, Purification, and Identification of Acr¹-[Taeg]₁₀-Lys-NH₂

Protected Acr¹-[Taeg]₁₀-Lys(ClZ)-BHA resin (99.1 mg, dry weight) wascleaved as described in Example 17(c) to yield 42.2 mg of crudematerial. The main peak at 25.3 minutes (eluted at 23.5 minutes ifinjected from an aqueous solution instead of the 10% aqueous acetic acidsolution) accounted for about 45% of the total absorbance. An 8.87 mgportion of the crude product was purified to give approximately 5.3 mgof >97% pure Acr¹-[Taeg]₁₀-Lys-NH₂. For (M+H)⁺ the calculated m/z valuewas 2850.8 and the measured m/z value was 2849.8.

(g) Cleavage and Purification of Acr¹-[Taeg]₁₅-Lys-NH₂

A 78.7 mg portion of protected Acr¹-[Taeg]₁₅-Lys(ClZ)-BHA resin (dryweight) was cleaved as described in Example 18 to yield 34.8 mg of crudematerial. The main peak at 23.5 minutes (about the same elution time ifinjected from an aqueous solution instead of the 10% aqueous acetic acidsolution) and a “dimer” at 28.2 minutes accounted for about 35% of thetotal absorbance. About 4.5 mg of the crude product was purified to giveapproximately 1.6 mg of presumably >95% pure Acr¹-[Taeg]₁₅-Lys-NH₂. Thiscompound could not be free of the “dimer” peak, which grew upon standingin aqueous acetic acid solution.

(h) Synthetic Protocol 2

(1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 3 mL, 3×1 minute and1×30 minutes; (2) washing with CH₂Cl₂, 3 mL, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 3 mL, 3×2 minutes; (4)washing with CH₂Cl₂, 3 mL, 6×1 minute, and drain for 1 minute; (5) 2-5mg sample of PNA-resin can be removed and dried thoroughly for aqualitative ninhydrin analysis; (6) for cycles 1 to 5 and cycles 10 to15 the coupling reaction was carried out by addition of 3.2 equiv. (0.18mmol, 100 mg) of BOC-Taeg-OPfp dissolved in 1 mL of CH₂Cl₂, followed byaddition of 0.5 mL of DMF (final concentration of pentafluorophenylester˜0.12 M). The coupling reaction was allowed to proceed for a total of12-24 h with shaking; cycles 5 to 10 employed an additional 0.12 M DCCcoupling of 0.12 M BOC-Taeg-OH in 1.5 mL of DMF/CH₂Cl₂ (1:2, v/v); (7)washing with DMF, 3 mL, 1×2 minutes; (8) washing with CH₂Cl₂, 3 mL, 4×1minute; (9) neutralization with DIEA/C₂Cl₂ (1:19, v/v), 3 mL, 2×2minutes; (10) washing with CH₂Cl₂, 3 mL, 6×1 minute; (11) 2-5 mg sampleof protected PNA-resin is removed for a qualitative ninhydrin test(after cycles 7, 10, and 15), and unreacted amino groups were blocked byacetylation with N-acetylimidazole in methylene chloride).

EXAMPLE 19

Improved Solid Phase Synthesis of H-[Taeg]₁₀-Lys-NH₂

The protected PNA was assembled onto an MBHA resin, using approximatelyhalf the loading of the BHA resin used in the previous examples.Furthermore, all cycles except one was followed by acetylation ofuncoupled amino groups. The following describes the synthesis in detail:

(a) Preparation of BOC-Lys(ClZ)—NH—CH(p-CH₃—C₆H₄)—C₆H₄ Resin (MBHAResin) With an Initial Substitution of 0.3 mmol/g

The desired substitution of BOC-Lys(ClZ)-MBHA resin was 0.25-0.30mmol/g. In order to get this value, 1.5 mmol of BOC-Lys(ClZ) was coupledto 5 g of neutralized and preswollen MBHA resin (determined byquantitative ninhydrin reaction to contain 0.64 mmol NH₂/g) using asingle “in situ” coupling (1.5 mmol of DCC) in 60 mL of CH₂Cl₂. Thereaction was carried out by shaking for 3 h in a manually operated, 225mL, standard, solid phase reaction vessel. Unreacted amino groups werethen blocked by acetylation with a mixture of aceticanhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 18 h. A quantitativeninhydrin reaction on the neutralized resin showed that only 0.00093mmol/g free amine remained (see Table I), i.e. 0.15% of the originalamino groups. The degree of substitution was estimated by deprotectionand ninhydrin analysis, and was found to be 0.32 mmol/g for theneutralized H-Lys(ClZ)-MBHA resin. This compares well with the maximumvalue of 0.28 mmol/g for a quantitative coupling of 0.30 mmolBOC-Lys(ClZ)/g resin (see Table II).

(b) Stepwise Assembly of BOC-[Taeg]₃-Lys(ClZ)-MBHA Resin

The entire batch of H-Lys(ClZ)-MBHA resin prepared in section (a) wasused directly (in the same reaction vessel) to assembleBOC-[Taeg]₃-Lys(ClZ)-MBHA resin by single couplings (“Synthetic Protocol3”) utilizing 2.5 equivalents of BOC-Taeg-OPfp in neat CH₂Cl₂. Thequantitative ninhydrin reaction was appplied throughout the synthesis(see Table II).

(c) Stepwise Assembly of BOC-[Taeg]₈-Lys(ClZ)-MBHA Resin

About 4.5 g of wet BOC-[Taeg]₃-Lys(ClZ)-MBHA resin (˜0.36 mmol growingchains, taken out of totally ˜19 g wet resin prepared in section (b))was placed in a 55 mL solid phase peptide synthesis (SPPS) reactionvessel. BOC-[Taeg]₈-Lys(ClZ)-MBHA resin was assembled by singlecouplings (“Synthetic Protocol 4”) utilizing 2.5 equivalents ofBOC-Taeg-OPfp in about 30% DMF/CH₂Cl₂. The progress of the synthesis wasmonitored at all stages by the quantitative ninhydrin reaction (seeTable II).

(d) Stepwise Assembly of BOC-[Taeg]₁₀-Lys(ClZ)-MBHA Resin

About 1 g of wet BOC-[Taeg]₈-Lys(ClZ)-MBHA resin (˜0.09 mmol growingchains, taken out of totally ˜4 g wet resin prepared in section (c)) wasplaced in a 20 mL SPPS reaction vessel. BOC-[Taeg]₁₀-Lys(ClZ)-MBHA resinwas assembled by the single-coupling protocol employed in the precedingsection utilizing 2.5 equivalents of BOC-Taeg-OPfp in about 30%DMF/CH₂Cl₂. The reaction volume was 3 mL (vigorous shaking). Thesynthesis was monitored by the quantitative ninhydrin reaction (seeTable II).

Remaining Free Amino Estimated Substitution After Groups After ExtentDeprotection (μmol/g) of Synthetic Residue (mmol/g) Single Coupling StepCoupled Measd. Theor. Coupling Acetyln. (%) “0” BOC- 0.32 0.28 0.93 Lys(ClZ) 1 BOC-Taeg 0.23 0.26 0.97 0.54 >99.9 2 BOC-Taeg 0.21 0.24 0.920.46 99.8 3 BOC-Taeg 0.19 0.23 1.00 0.57 99.7 4 BOC-Taeg 0.18 0.21 1.8599.3 5 BOC-Taeg 0.17 0.20 2.01 0.19 99.9 6 BOC-Taeg 0.15 0.19 1.69 0.1099.0 7 BOC-TAeg 0.11 0.18 1.11 0.66 99.1 8 BOC-Taeg 0.12 0.17 1.82 0.4499.0 9 BOC-Taeg 0.10 0.17 5.63 0.56 94.8 10  BOC-Taeg 0.11 0.16 1.540.67 99.1

(e) Synthesis of Ac-[Taeg]₁₀-Lys(ClZ)-MBHA Resin

Following deprotection of a portion of Boc-[Taeg]₁₀-Lys(ClZ)-MBHA resin(estimated dry weight is about 45 mg), the resin was next acetylatedquantitatively with a 2 mL mixture of acetic anhydride/pyridine/CH₂Cl₂(1:1:2, v/v/v) for 2 h in a 3 mL solid phase reaction vessel.

(f) Cleavage, Purification, and Identification of H-[Taeg]₁₀-Lys-NH₂

A portion of protected Boc-[Taeg]₁₀-Lys(ClZ)-BHA resin was treated asdescribed in Example 17(c) to yield about 24 mg of crude material uponHF cleavage of 76 mg dry H-[Taeg]₅-Lys(ClZ)-BHA resin. The main peak at15.2 minutes which includes impurities such as deletion peptides andvarious byproducts) accounted for about 78% of the total absorbance. Themain peak also accounted for about 88% of the “main peak plus deletionpeaks” absorbance, which is in good agreement with the overall estimatedcoupling yield of 90.1% obtained by summarizing the individual couplingyields in Table II. A 7.2 mg portion of the crude product was purifiedfrom two batches by use of a semi-preparative reverse-phase column,(collecting the main peak in a beaker cooled with dry ice/2-propanol).Each contained 3.6 mg in 1 mL of H₂O. The frozen solution waslyophilized directly (without prior removal of acetonitrile on a speedvac) to give 4.2 mg of 82% pure H-[Taeg]₁₀-Lys-NH₂.

(g) Cleavage, Purification, and Identification of Ac-[Taeg]₁₀-Lys-NH₂

A 400.0 mg portion of protected Ac-[Taeg]₁₀-Lys(ClZ)-BHA resin (dryweight) was cleaved as described in Example 17(c), except for the TFAtreatment to yield 11.9 mg of crude material. The main peak at 15.8minutes accounted for about 75% of the total absorbance. A 4.8 mgportion of the crude product was purified to give approximately 3.5 mgof >95% pure Ac-[Taeg]₁₀-Lys-NH₂. For (M+H)⁺ the calculated m/zvalue=2849.8 and the measured m/z value=2848.8.

(h) Synthetic Protocol 3

(1) Boc-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 100 mL, 3×1 minute and1×30 minutes; (2) washing with CH₂Cl₂, 100 mL, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 100 mL, 3×2 minutes; (4)washing with CH₂Cl₂, 100 mL, 6×1 minute, and drain for 1 minute; (5) 2-5mg sample of PNA-resin is removed and dried thoroughly for aquantitative ninhydrin analysis to determine the substitution; (6)addition of 2.5 equiv. (3.75 mmol; 2.064 g) BocTaeg-OPfp dissolved in 35mL CH₂Cl₂ (final concentration of pentafluorophenylester ˜0.1 M); thecoupling reaction was allowed to proceed for a total of 20-24 h withshaking; (7) washing with DMF, 100 mL, 1×2 minutes (to removeprecipitate of BOC-Taeg-OH); (8) washing with CH₂Cl₂, 100 mL, 4×1minute; (9) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 100 mL, 2×2minute; (10) washing with CH₂Cl₂, 100 mL, 6×1 minute; (11) 2-5 mg sampleof protected PNA-resin was removed for a rapid qualitative ninhydrintest and a further 2-5 mg is dried thoroughly for a quantitativeninhydrin analysis to determine the extent of coupling; (12) blocking ofunreacted amino groups by acetylation with a 100 mL mixture of aceticanhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h; (13) washing withCH₂Cl₂, 100 mL 6×1 minute; (14) 2×2-5 mg samples of protected PNA-resinwere removed, neutralized with DIEA/CH₂Cl₂ (1:19, v/v) and washed withCH₂Cl₂ for qualitative and quantitative ninhydrin analyses.

(i) Synthetic Protocol 4

(1) Boc-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 25 mL, 3×1 min and 1×30minutes; (2) washing with CH₂Cl₂, 25 mL, 6×1 minute; (3) neutralizationwith DIEA/CH₂Cl₂ (1:19, v/v), 25 mL, 3×2 minutes; (4) washing withCH₂Cl₂, 25 mL, 6×1 minute, and drain for 1 minute; (5) 2-5 mg sample ofPNA-resin was removed and dried thoroughly for a quantitative ninhydrinanalysis to determine the substitution; (6) addition of 2.5 equiv. (0.92mmol; 0.506 g) BocTaeg-OPfp dissolved in 6 mL CH₂Cl₂ followed byaddition of 3 mL DMF (final concentration of pentafluorophenylester ˜0.1M); the coupling reaction was allowed to proceed for a total of 20-24hrs with shaking; (7) washing with DMF, 25 mL, 1×2 minutes; (8) washingwith CH₂Cl₂, 25 mL, 4×1 minute; (9) neutralization with DIEA/CH₂Cl₂(1:19, v/v), 25 mL, 2×2 minutes; (10) washing with CH₂Cl₂, 25 mL, 6×1minute; (11) 2-5 of protected PNA-resin was removed for a rapidqualitative ninhydrin test and a further 2-5 mg is dried thoroughly fora quantitative ninhydrin analysis to determine the extent of coupling;(12) blocking of unreacted amino groups by acetylation with a 25 mLmixture of acetic anhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h(except after the first cycle); (13) washing with CH₂Cl₂, 25 mL, 6×1minute; (14) 2×2-5 mg samples of protected PNA-resin are taken out,neutralized with DIEA/CH₂Cl₂ (1:19, v/v) and washed with CH₂Cl₂ forqualitative and quantitative ninhydrin analyses.

EXAMPLE 20 Synthesis of N-benzyloxycarbonyl-N-′(BOC-aminoethyl)glycine

Aminoethyl glycine (52.86 g, 0.447 mol) was dissolved in water (900 mL)and dioxane (900 mL) was added. The pH was adjusted to 11.2 with 2NNaOH. While the pH was kept at 11.2, tert-butyl-p-nitrophenyl carbonate(128.4 g, 0.537 mol) was dissolved in dioxane (720 mL) and addeddropwise over the course of 2 hours. The pH was kept at 11.2 for atleast three more hours and then allowed to stand overnight, withstirring. The yellow solution was cooled to 0° C. and the pH wasadjusted to 3.5 with 2 N HCl. The mixture was washed with chloroform(4×100 mL), and the pH of the aqueous phase was readjusted to 9.5 with 2N NaOH at 0° C. Benzyloxycarbonyl chloride (73.5 mL, 0.515 mol) wasadded over half an hour, while the pH was kept at 9.5 with 2 N NaOH. ThepH was adjusted frequently over the next 4 hours, and the solution wasallowed to stand overnight, with stirring. On the following day thesolution was washed with ether (3×600 mL) and the pH of the solution wasafterwards adjusted to 1.5 with 2 N HCl at 0° C. The title compound wasisolated by extraction with ethyl acetate (5×1000 mL). The ethyl acetatesolution was dried over magnesium sulfate and evaporated to dryness, invacuo. This afforded 138 g of the product, which was dissolved in ether(300 mL) and precipitated by the addition of petroleum ether (1800 mL).Yield 124.7 g (79%). M.p. 64.5-85° C. Anal. for C₁₇H₂₄N₂O₆ found(calc.)C, 58.40(57.94); H, 7.02(6.86); N, 7.94(7.95). ¹H-NMR (250 MHz, CDCl₃)7.33 & 7.32 (5H, Ph); 5.15 & 5.12 (2H, PhCH ₂); 4.03 & 4.01 (2H, NC₂ HC₂O H); 3.46 (b, 2H, BOC—NHCH₂CH ₂); 3.28 (b, 2H BOC—NHCH ₂CH₂); 1.43 &1.40 (9H t-Bu). HPLC (260 nm) 20.71 min. (80.2%) and 21.57 min. (19.8%).The UV-spectra (200 nm-300 nm) are identical, indicating that the minorpeak consists of Bis-Z-AEG.

EXAMPLE 21 Synthesis of N′-BOC-aminoethylglycine ethyl ester

N-Benzyloxycarbonyl-N′-(BOC-aminoethyl)glycine (60 g, 0.170 mol) andN,N-dimethyl-4-aminopyridine (6 g) were dissolved in absolute ethanol(500 mL), and cooled to 0° C. before the addition of DCC (42.2 g, 0.204mol). The ice bath was removed after 5 minutes and stirring wascontinued for 2 more hours. The precipitated DCU (32.5 g, dried) wasremoved by filtration and washed with ether (3×100 mL). The combinedfiltrate was washed successively with diluted potassium hydrogen sulfate(2×400 mL), diluted sodium hydrogencarbonate (2×400 mL) and saturatedsodium chloride (1×400 mL). The organic phase was filtered, then driedover magnesium sulfate, and evaporated to dryness, in vacuo, whichyielded 66.1 g of an oily substance which contained some DCU.

The oil was dissolved in absolute ethanol (600 mL) and was added 10%palladium on carbon (6.6 g) was added. The solution was hydrogenated atatmospheric pressure, where the reservoir was filled with 2 N sodiumhydroxide. After 4 hours, 3.3 L was consumed out of the theoretical 4.2L. The reaction mixture was filtered through celite and evaporated todryness, in vacuo, affording 39.5 g (94%) of an oily substance. A 13 gportion of the oily substance was purified by silica gel (SiO₂, 600 g)chromatography. After elution with 300 mL of 20% petroleum ether inmethylene chloride, the title compound was eluted with 1700 mL of 5%methanol in methylene chloride. The solvent was removed from thefractions with satisfactory purity, in vacuo and the yield was 8.49 g.Alternatively 10 g of the crude material was purified by Kugel Rohrdistillation. ¹H-NMR (250 MHz, CD₃OD); 4.77 (b. s, NH); 4.18 (q, 2H,MeCH ₂—); 3.38 (s, 2H, NCH ₂CO₂Et); 3.16 (t, 2H, BOC—NHCH ₂CH₂); 2.68(t, 2H, BOC—NHCH₂CH ₂); 1.43 (s, 9H, t-Bu) and 1.26 (t, 3H, CH₃) ¹³C-NMR171.4 (COEt); 156.6 (CO); 78.3 ((CH₃)₃ C); 59.9 (CH₂); 49.0 (CH₂); 48.1(CH₂); 39.0 (CH₂); 26.9 (CH₂) and 12.6 (CH₃).

EXAMPLE 22 Synthesis of N′-BOC-aminoethylglycine methyl ester

The above procedure was used, with methanol being substituted forethanol. The final product was purified by column purification.

EXAMPLE 23 Synthesis of 1-(BOC-aeg)thymine ethyl ester

N′-BOC-aminoethylglycine ethyl ester (13.5 g, 54.8 mmol), DhbtOH (9.84g, 60.3 mmol) and 1-carboxymethyl thymine (11.1 g, 60.3 mmol) weredissolved in DMF (210 mL). Methylene chloride (210 mL) was added. Thesolution was cooled to 0° C. in an ethanol/ice bath and DCC (13.6 g,65.8 mmol) was added. The ice bath was removed after 1 hour and stirringwas continued for another 2 hours at ambient temperature. Theprecipitated DCU was removed by filtration and washed twice withmethylene chloride (2×75 mL). To the combined filtrate was added moremethylene chloride (650 mL). The solution was washed successively withdiluted sodium hydrogen carbonate (3×500 mL), diluted potassium hydrogensulfate (2×500 mL), and saturated sodium chloride (1×500 mL). Some ofthe precipitate was removed from the organic phase by filtration, Theorganic phase was dried over magnesium sulfate and evaporated todryness, in vacuo. The oily residue was dissolved in methylene chloride(150 mL), filtered, and the title compound was precipitated by theaddition of petroleum ether (350 mL) at 0° C. The methylenechloride/petroleum ether procedure was repeated once. This afforded 16 g(71%) of a material which was more than 99% pure by HPLC.

EXAMPLE 24 Synthesis of 1-(BOC-aeg)thymine

The material from above was suspended in THF (194 mL, gives a 0.2 Msolution), and 1 M aqueous lithium hydroxide (116 mL) was added. Themixture was stirred for 45 minutes at ambient temperature and thenfiltered to remove residual DCU. Water (40 mL) was added to the solutionwhich was then washed with methylene chloride (300 mL). Additional water(30 mL) was added, and the alkaline solution was washed once more withmethylene chloride (150 mL). The aqueous solution was cooled to 0° C.and the pH was adjusted to 2 by the dropwise addition of 1 N HCl (approx110 mL). The title compound was extracted with ethyl acetate (9×200 mL),the combined extracts were dried over magnesium sulfate and wereevaporated to dryness, in vacuo. The residue was evaporated once frommethanol, which after drying overnight afforded a colorless glassysolid. Yield: 9.57 g (64%). HPLC>98% R_(T)=14.8 minutes. Anal. forC₁₆H₂₄N₄O₇.0.25 H₂O Found (calc.) C, 49.29(49.42); H, 6.52(6.35); N,14.11(14.41). Due to the limited rotation around the secondary amide,several of the signals were doubled in the ratio 2:1 (indicated in thelist by mj. for major and mi. for minor). ¹H-NMR (250 MHz, DMSO-d₆) δ:12.75 (bs, 1H, CO₂H); 11.28 (s, 1H, mj, imide NH); 11.26 (s, 1H, mi,imide NH); 7.30 (s, 1H, mj, T H-6); 7.26 (s, 1H, mi, T H-6); 6.92 (bt,1H, mj, BOC—NH); 6.73 (bt, 1H, mi, BOC—NH); 4.64 (s, 2H, mj, CH ₂CON);4.46 (s, 2H, mj, CH ₂CON); 4.19 (s, 2H, mi, CH ₂CO₂H); 3.97 (s, 2H, mj,CH ₂CO₂H); 3.63-3.01 (unresolved m, includes water, CH ₂CH ₂); 1.75 (s,3H, CH ₃) and 1.38 (s, 9H, t-Bu).

EXAMPLE 25 Synthesis of N⁴-benzyloxycarbonyl-1-(BOC-aeg)cytosine

N′-BOC-aminoethyl glycine ethyl ester (5 g, 20.3 mmol), DhbtOH (3.64 g,22.3 mmol) and N⁴-benzyloxycarbonyl-1-carboxymethyl cytosine (6.77 g,22.3 mmol) were suspended in DMF (100 mL). Methylene chloride (100 mL)was added. The solution was cooled to 0° C. and DCC (5.03 g, 24.4 mmol)was added. The ice bath was removed after 2 h and stirring was continuedfor another hour at ambient temperature. The reaction mixture then wasevaporated to dryness, in vacuo. The residue was suspended in ether (100mL) and stirred vigorously for 30 minutes. The solid material wasisolated by filtration and the ether wash procedure was repeated twice.The material was then stirred vigorously for 15 minutes with dilutesodium hydrogencarbonate (aprox. 4% solution, 100 mL), filtered andwashed with water. This procedure was then repeated once, which afterdrying left 17 g of yellowish solid material. The solid was thenrefluxed with dioxane (200 mL) and filtered while hot. After cooling,water (200 mL) was added. The precipitated material was isolated byfiltration, washed with water, and dried. According to HPLC (observingat 260 nm) this material has a purity higher than 99%, besides the DCU.The ester was then suspended in THF (100 ml), cooled to 0° C., and 1 NLiOH (61 mL) was added. After stirring for 15 minutes, the mixture wasfiltered and the filtrate was washed with methylene chloride (2×150 mL).The alkaline solution then was cooled to 0° C. and the pH was adjustedto 2.0 with 1 N HCl. The title compound was isolated by filtration andwas washed once with water, leaving 11.3 g of a white powder afterdrying. The material was suspended in methylene chloride (300 mL) andpetroleum ether (300 mL) was added. Filtration and wash afforded 7.1 g(69%) after drying. HPLC showed a purity of 99% R_(T)=19.5 minutes, anda minor impurity at 12.6 minutes (approx. 1%) most likely theZ-deprotected monomer. Anal. for C₂₃H₂₉N₅O₈ found(calc.) C,54.16(54.87); H, 5.76(5.81) and N, 13.65(13.91). ¹H-NMR (250 MHzDMSO-d₆). 10.78 (bs, 1H, CO₂ H); 7.88 (2 overlapping doublets, 1H, CytH-5); 7.41-7.32 (m, 5H, Ph); 7.01 (2 overlapping doublets, 1H, Cyt H-6);6.94 & 6.78 (unresolved triplets, 1H, BOC—NH); 5.19 (s, 2H, PhCH ₂);4.81 & 4.62 (s, 2H, CH ₂CON); 4.17 & 3.98 (s, 2H, CH ₂CO₂H); 3.42-3.03(m, includes water, CH ₂CH ₂) and 1.38 & 1.37 (s, 9H, ^(t)-Bu). ¹³C-NMR.150.88; 128.52; 128.18; 127.96; 93.90; 66.53; 49.58 and 28.22. IR:Frequency in cm⁻¹ (intensity). 3423 (26.4), 3035 (53.2), 2978(41.4),1736(17.3), 1658(3.8), 1563(23.0), 1501(6.8) and 1456 (26.4).

EXAMPLE 26 Synthesis of 9-carboxymethyladenine ethyl ester

Adenine (10 g, 74 mmol) and potassium carbonate (10.29 g, 74 mmol) weresuspended in DMF and ethyl bromoacetate (8.24 mL, 74 mmol) was added.The suspension was stirred for 2.5 h under nitrogen at room temperatureand then filtered. The solid residue was washed three times with DMF (10mL). The combined filtrate was evaporated to dryness, in vacuo. Water(200 mL) was added to the yellowish-orange solid material and the pHadjusted to 6 with 4 N HCl. After stirring at 0° C. for 10 minutes, thesolid was filtered off, washed with water, and recrystallized from 96%ethanol (150 mL). The title compound was isolated by filtration andwashed thoroughly with ether. Yield: 3.4 g (20%). M.p. 215.5-220° C.Anal. for C₉H₁₁N₅O₂ found(calc.): C, 48.86(48.65); H, 5.01(4.91); N,31.66(31.42). ¹H-NMR (250 MHz; DMSO-d₆): 7.25 (bs, 2H NH₂), 5.06 (s, 2H,NCH₂), 4.17 (q, 2H, J=7.11 Hz, OCH₂) and 1.21 (t, 3H, J=7.13 Hz, NCH₂).¹³C-NMR. 152.70, 141.30, 61.41, 43.97 and 14.07. FAB-MS. 222 (MH+). IR:Frequency in cm⁻¹ (intensity). 3855 (54.3), 3274(10.4), 3246(14.0),3117(5.3), 2989(22.3), 2940(33.9), 2876(43.4), 2753(49.0), 2346(56.1),2106(57.1), 1899(55.7), 1762(14.2), 1742(14.2), 1742(1.0), 1671(1.8),1644(10.9), 1606(0.6), 1582(7.1), 1522(43.8), 1477(7.2), 1445(35.8) and1422(8.6). The position of alkylation was verified by X-raycrystallography on crystals, which were obtained by recrystallizationfrom 96% ethanol.

Alternatively, 9-carboxymethyladenine ethyl ester can be prepared by thefollowing procedure. To a suspension of adenine (50 g, 0.37 mol) in DMF(1100 mL) in 2 L three-necked flask equipped with a nitrogen inlet, amechanical stirrer and a dropping funnel, was added 16.4 g (0.407 mol)of hexane-washed sodium hydride-mineral oil dispersion. The mixture wasstirred vigorously for 2 hours, after which ethyl bromacetate (75 mL,0.67 mol) was added dropwise over the course of 3 hours. The mixture wasstirred for one additional hour, after which tlc indicated completeconversion of adenine. The mixture was evaporated to dryness at 1 mm Hgand water (500 mL) was added to the oily residue which causedcrystallization of the title compound. The solid was recrystallised from96% ethanol (600 mL). Yield (after drying): 53.7 g (65.6%). HPLC (215nm) purity>99.5%.

EXAMPLE 27 Synthesis of N⁶-benzyloxycarbonyl-9-carboxymethyladenineethyl ester

9-Carboxymethyladenine ethyl ester (3.4 g, 15.4 mmol) was dissolved indry DMF (50 mL) by gentle heating, cooled to 20° C., and added to asolution of N-ethyl-benzyloxycarbonylimidazole tetrafluoroborate (62mmol) in methylene chloride (50 mL) over a period of 15 minutes in anice bath. Some precipitation was observed. The ice bath was removed andthe solution was stirred overnight. The reaction mixture was treatedwith saturated sodium hydrogen carbonate (100 mL). After stirring for 10minutes, the phases were separated and the organic phase was washedsuccessively with one volume of water, dilute potassium hydrogen sulfate(twice), and with saturated sodium chloride. The solution was dried overmagnesium sulfate and evaporated to dryness, in vacuo, which afforded 11g of an oily material. The material was dissolved in methylene chloride(25 mL), cooled to 0° C., and precipitated with petroleumeum ether (50mL). This procedure was repeated once to give 3.45 g (63%) of the titlecompound. M.p. 132-35° C. Analysis for C₁₇H₁₇N₅O₄ found (calc.): C,56.95(57.46); H, 4.71(4.82); N, 19.35(19.71). ¹H-NMR (250 MHz; CDCl₃):8.77 (s, 1H, H-2 or H-8); 7.99 (s, 1H, H-2 or H-8); 7.45-7.26 (m, 5H,Ph); 5.31 (s, 2H, N—CH ₂); 4.96 (s, 2H, Ph-CH ₂); 4.27 (q, 2H, J=7.15Hz, CH ₂CH₃) and 1.30 (t, 3H, J=7.15 Hz, CH₂CH₃). ¹³C-NMR: 153.09;143.11; 128.66; 67.84; 62.51; 44.24 and 14.09. FAB-MS: 356 (MH+) and 312(MH+-CO₂). IR: frequency in cm⁻¹ (intensity). 3423 (52.1); 3182 (52.8);3115(52.1); 3031(47.9); 2981(38.6); 1747(1.1); 1617(4.8); 15.87(8.4);1552(25.2); 1511(45.2); 1492(37.9); 1465(14.0) and 1413(37.3).

EXAMPLE 28 Synthesis of N⁶-benzyloxycarbonyl-9-carboxymethyladenine

N⁶-Benzyloxycarbonyl-9-carboxymethyladenine ethylester (3.2 g, 9.01mmol) was mixed with methanol (50 mL) cooled to 0° C. Sodium hydroxidesolution (2 N, 50 mL) was added, whereby the material quickly dissolved.After 30 minutes at 0° C., the alkaline solution was washed withmethylene chloride (2×50 mL). The pH of the aqueous solution wasadjusted to 1 with 4 N HCl at 0° C., whereby the title compoundprecipitated. The yield after filtration, washing with water, and dryingwas 3.08 g (104%). The product contained salt, and the elementalanalysis reflected that. Anal. for C₁₅H₁₃N₅O₄ found(calc.): C,46.32(55.05); H, 4.24(4.00); N, 18.10(21.40); and C/N, 2.57(2.56).¹H-NMR(250 MHz; DMSO-d₆): 8.70 (s, 2H, H-2 and H-8); 7.50-7.35 (m, 5H,Ph); 5.27 (s, 2H, N—CH ₂); and 5.15 (s, 2H, Ph-CH ₂). ¹³C-NMR 168.77,152.54, 151.36, 148.75, 145.13, 128.51, 128.17, 127.98, 66.76 and44.67.IR (KBr) 3484(18.3); 3109(15.9); 3087(15.0); 2966(17.1);2927(19.9); 2383(53.8) 1960(62.7); 1739(2.5); 1688(5.2); 1655(0.9);1594(11.7); 1560(12.3); 1530(26.3); 1499(30.5); 1475(10.4); 1455(14.0);1429(24.5) and 1411(23.6). FAB-MS: 328 (MH+) and 284 (MH+-CO₂). HPLC(215 nm, 260 nm) in system 1:15.18 min, minor impurities all less than2%.

EXAMPLE 29 Synthesis of N⁶-benzyloxycarbonyl-1-(BOC-aeg)adenine ethylester

N′-BOC-aminoethylglycine ethyl ester (2 g, 8.12 mmol), DhbtOH (1.46 g,8.93 mmol) and N⁶-benzyloxycarbonyl-9-carboxymethyl adenine (2.92 g,8.93 mmol) were dissolved in DMF (15 mL). Methylene chloride (15 mL)then was added. The solution was cooled to 0° C. in an ethanol/ice bath.DCC (2.01 g, 9.74 mmol) was added. The ice bath was removed after 2.5 hand stirring was continued for another 1.5 hour at ambient temperature.The precipitated DCU was removed by filtration and washed once with DMF(15 mL), and twice with methylene chloride (2×15 mL). To the combinedfiltrate was added more methylene chloride (100 mL). The solution waswashed successively with dilute sodium hydrogen carbonate (2×100 mL),dilute potassium hydrogen sulfate (2×100 mL), and saturated sodiumchloride (1×100 mL). The organic phase was evaporated to dryness, invacuo, which afforded 3.28 g (73%) of a yellowish oily substance. HPLCof the raw product showed a purity of only 66% with several impurities,both more and less polar than the main peak. The oil was dissolved inabsolute ethanol (50 mL) and activated carbon was added. After stirringfor 5 minutes, the solution was filtered. The filtrate was mixed withwater (30 mL) and was allowed to stir overnight. The next day, the whiteprecipitate was removed by filtration, washed with water, and dried,affording 1.16 g (26%) of a material with a purity higher than 98% byHPLC. Addition of water to the mother liquor afforded another 0.53 g ofthe product with a purity of approx. 95%. Anal. for C₂₆H₃₃N₇O₇.H₂Ofound(calc.) C, 55.01(54.44; H, 6.85(6.15); and N, 16.47(17.09). ¹H-NMR(250 MHz, CDCl₃) 8.74 (s, 1H, Ade H-2); 8.18 (b, s, 1H, ZNH); 8.10 &8.04 (s, 1H, H-8); 7.46-7.34 (m, 5H, Ph); 5.63 (unres. t, 1H, BOC—NH);5.30 (s, 2H, PhCH₂); 5.16 & 5.00 (s, 2H, CH ₂CON); 4.29 & 4.06 (s, 2H,CH ₂CO₂H); 4.20 (q, 2H, OCH ₂CH₃); 3.67-3.29 (m, 4H, CH ₂CH ₂); 1.42 (s9H, t-Bu) and 1.27 (t, 3H, OCH₂CH ₃). The spectrum shows traces ofethanol and DCU.

EXAMPLE 30 Synthesis of N⁶-benzyloxycarbonyl-1-(BOC-aeg)adenine

N⁶-Benzyloxycarbonyl-1-(BOC-aeg)adenine ethyl ester (1.48 g, 2.66 mmol)was suspended in THF (13 mL) and the mixture was cooled to 0° C. Lithiumhydroxide (8 mL, 1 N) was added. After 15 minutes of stirring, thereaction mixture was filtered, extra water (25 mL) was added, and thesolution was washed with methylene chloride (2×25 mL). The pH of theaqueous solution was adjusted to 2 with 1 N HCl. The precipitate wasisolated by filtration, washed with water, and dried, affording 0.82 g(58%) of the product. The product was additionally precipitated twicewith methylene chloride/petroleum ether. Yield (after drying): 0.77 g(55%). M.p. 119° C. (decomp.). Anal. for C₂₄H₂₉N₇O₇.H₂O found(calc.) C,53.32(52.84); H, 5.71(5.73); N, 17.68(17.97). FAB-MS. 528.5 (MH+).¹H-NMR (250 MHz, DMSO-d₆). 12.75 (very b, 1H, CO₂H); 10.65 (b. s, 1H,ZNH); 8.59 (d, 1H, J=2.14 Hz, Ade H-2); 8.31 (s, 1H, Ade H-8); 7.49-7.31(m, 5H, Ph); 7.03 & 6.75 (unresol. t, 1H, BOC—NH); 5.33 & 5.16 (s, 2H,CH₂CON); 5.22 (s, 2H, PhCH ₂); 4.34-3.99 (s, 2H, CH₂CO₂H); 3.54-3.03(m's, includes water, CH ₂CH ₂) and 1.39 & 1.37 (s, 9H, t-Bu). ¹³C-NMR.170.4; 166.6; 152.3; 151.5; 149.5; 145.2; 128.5; 128.0; 127.9; 66.32;47.63; 47.03; 43.87 and 28.24.

EXAMPLE 31 Synthesis of 2-amino-6-chloro-9-carboxymethylpurine

To a suspension of 2-amino-6-chloropurine (5.02 g, 29.6 mmol) andpotassium carbonate (12.91 g, 93.5 mmol) in DMF (50 mL) was addedbromoacetic acid (4.7 g, 22.8 mmol). The mixture was stirred vigorouslyfor 20 h under nitrogen. Water (150 mL) was added and the solution wasfiltered through celite to give a clear yellow solution. The solutionwas acidified to a pH of 3 with 4 N hydrochloric acid. The precipitatewas filtered and dried, in vacuo, over sicapent. Yield: 3.02 g (44.8%).¹H-NMR(DMSO-d₆) δ: 4.88 ppm (s, 2H); 6.95 (s, 2H); 8.10 (s, 1H).

EXAMPLE 32 Synthesis of 2-amino-6-benzyloxy-9-carboxymethylpurine

Sodium (2 g, 87 mmol) was dissolved in benzyl alcohol (20 mL) and heatedto 130° C. for 2 h. After cooling to 0° C., a solution of2-amino-6-chloro-9-carboxymethylpurine (4.05 g, 18 mmol) in DMF (85 mL)was slowly added, and the resulting suspension stirred overnight at 20°C. Sodium hydroxide solution (1 N, 100 mL) was added and the clearsolution was washed with ethyl acetate (3×100 mL). The water phase wasthen acidified to a pH of 3 with 4 N hydrochloric acid. The precipitatewas taken up in ethyl acetate (200 mL), and the water phase wasextracted with ethyl acetate (2×100 mL). The combined organic phaseswere washed with saturated sodium chloride solution (2×75 mL), driedwith anhydrous sodium sulfate, and evaporated to dryness, in vacuo. Theresidue was recrystallized from ethanol (300 mL). Yield, after drying invacuo, over sicapent: 2.76 g (52%). M.p. 159-65° C. Anal. (calc.;found): C,(56.18; 55.97); H,(4.38; 4.32); N,(23.4; 23.10). ¹H-NMR(DMSO-d₆) δ: 4.82 (s, 2H); 5.51 (s, 2H); 6.45 (s, 2H); 7.45 (m, 5H);7.82 (s, 1H).

EXAMPLE 33 Synthesis ofN-([2-amino-6-benzyloxy-purine-9-yl]-acetyl)-N-(2-BOC-aminoethyl)glycine[BOC-Gaeg-OH monomer]

2-Amino-1-benzyloxy-9-carboxymethyl-purine (0.5 g, 1.67 mmol),methyl-N(2-[tert-butoxycarbonylamino]ethyl)-glycinate (0.65 g, 2.8mmol), diisopropylethyl amine (0.54 g, 4.19 mmol), andbromo-tris-pyrrolidino-phosphonium-hexafluoro-phosphate (PyBroP®) (0.798g, 1.71 mmol) were stirred in DMF (2 mL) for 4 h. The clear solution waspoured into an ice-cooled solution of sodium hydrogen carbonate (1 N, 40mL) and extracted with ethyl acetate (3×40 mL). The organic layer waswashed with potassium hydrogen sulfate solution (1 N, 2×40 mL), sodiumhydrogen carbonate (1 N, 1×40 mL) and saturated sodium chloride solution(60 mL). After drying with anhydrous sodium sulfate and evaporation invacuo, the solid residue was recrystallized from 2:1 ethylacetate/hexane (20 mL) to give the methyl ester in 63% yield. (MS-FAB514 (M+1). Hydrolysis was accomplished by dissolving the ester in 1:2ethanol/water (30 mL) containing concentrated sodium hydroxide (1 mL).After stirring for 2 h, the solution was filtered and acidified to a pHof 3, by the addition of 4 N hydrochloric acid. The title compound wasobtained by filtration. Yield: 370 mg (72% for the hydrolysis). Purityby HPLC was more than 99%. Due to the limited rotation around thesecondary amide several of the signals were doubled in the ratio 2:1(indicated in the list by mj for major and mi for minor). ¹H-NMR(250,MHz, DMSO-d₆) δ: 1.4 (s, 9H); 3.2 (m, 2H); 3.6 (m, 2H); 4.1 (s, mj,CONRCH ₂COOH); 4.4 (s, mi, CONRCH ₂COOH); 5.0 (s, mi, Gua-CH ₂CO—); 5.2(s, mj, Gua-CH ₂CO); 5.6 (s, 2H); 6.5 (s, 2H); 6.9 (m, mi, BOC—NH); 7.1(m, mj, BOC—NH); 7.5 (m, 3H); 7.8 (s, 1H); 12,8 (s, 1H). ¹³C-NMR.170.95; 170.52; 167.29; 166.85; 160.03; 159.78; 155.84; 154.87; 140.63;136.76; 128.49; 128.10; 113.04; 78.19; 77.86; 66.95; 49.22; 47.70;46.94; 45.96; 43.62; 43.31 and 28.25.

EXAMPLE 34 Synthesis of 3-BOC-amino-1,2-propanediol

3-Amino-1,2-propanediol (1 equivalent, 40 g, 0.44 mol) was dissolved inwater (1000 mL) and cooled to 0° C. Di-tert-butyl dicarbonate (1.2equivalents, 115 g, 0.526 mol) was added in one portion. The reactionmixture was heated to room temperature on a water bath during stirring.The pH was maintained at 10.5 with a solution of sodium hydroxide (1equivalent, 17.56 g, 0.44 mol) in water (120 mL). When the addition ofaqueous sodium hydroxide was completed, the reaction mixture was stirredovernight at room temperature. Subsequently, ethyl acetate (750 mL) wasadded to the reaction mixture, followed by cooling to 0° C. The pH wasadjusted to 2.5 with 4 N sulphuric acid with vigorous stirring. Thephases were separated and the water phase was washed with additionalethyl acetate (6×350 mL). The volume of the organic phase was reduced to900 mL by evaporation under reduced pressure. The organic phase was thenwashed with a saturated aqueous solution of potassium hydrogen sulfitediluted to twice its volume (1×1000 mL) and with saturated aqueoussodium chloride (1×500 mL). The organic phase was dried (MgSO₄) andevaporated under reduced pressure to yield 50.12 g (60%) of the titlecompound. The product could be solidified by evaporation from methylenechloride and subsequent freezing. ¹H-NMR (CDCl₃/TMS) δ: 1.43 (s, 9H,Me₃C), 3.25 (m, 2H, CH₂), 3.57 (m, 2H, CH₂), 3.73 (m, 1H, CH). ¹³C-NMR(CDCl₃/TMS) ppm: 28.2 (Me₃C), 42.6 (CH₂), 63.5, 71.1 (CH₂OH, CHOH), 79.5(Me₃C), 157.0 (C═O).

EXAMPLE 35 Synthesis of 2-(BOC-amino)ethyl-L-alanine methyl ester

3-BOC-amino-1,2-propanediol (1 equivalent, 20.76 g, 0.109 mol) wassuspended in water (150 mL). Potassium periodate (1 equivalent, 24.97 g,0.109 mol) was added and the reaction mixture was stirred for 2 h atroom temperature under nitrogen. The reaction mixture was filtered andthe water phase extracted with chloroform (6×250 mL). The organic phasewas dried (MgSO₄) and evaporated to afford an almost quantitative yieldof BOC-aminoacetaldehyde as a colorless oil, which was used withoutfurther purification in the following procedure.

Palladium on carbon (10%, 0.8 g) was added to MEOH (250 mL) undernitrogen with cooling (0° C.) and vigorous stirring. Anhydrous sodiumacetate (2 equivalents, 4.49 g, 54.7 mmol) and L-alanine methyl ester,hydrochloride (1 equivalent, 3.82 g, 27.4 mmol) were added.BOC-aminoacetaldehyde (4.79 g, 30.1 mmol, 1.1 eqv) was dissolved in MeOH(150 mL) and added to the reaction mixture. The reaction mixture washydrogenated at atmospheric pressure and room temperature until hydrogenuptake had ceased. The reaction mixture was filtered through celite,which was washed with additional MEOH. The MeOH was removed underreduced pressure. The residue was suspended in water (150 mL) and the pHadjusted to 8 by dropwise addition of 0.5 N NaOH with vigorous stirring.The water phase was extracted with methylene chloride (4×250 mL). Theorganic phase was dried (MgSO₄), filtered through celite, and evaporatedunder reduced pressure to yield 6.36 g (94%) of the title compound as aclear, pale yellow oil. MS (FAB-MS): m/z (%)=247 (100, M+1, 191 (90),147 (18). ¹H-NMR (250 MHz, CDCl₃) δ: 1.18 (d, J=7.0 Hz, 3H, Me), 1.36(s, 9H, Me₃C), 1.89 (b, 1H, NH), 2.51 (m, 1H, CH₂), 2.66 (m, 1H, CH₂),3.10 (m, 2H, CH₂), 3.27 (q, J=7.0 Hz, 1N, CH), 3.64 (s, 3H, OMe), 5.06(b, 1H, carbamate NH). ¹³C-NMR (ppm): 18.8 (Me), 28.2 (Me₃C), 40.1, 47.0(CH₂), 51.6 (OMe), 56.0 (CH), 155.8 (carbamate C═O), 175.8 (ester C═O).

EXAMPLE 36 Synthesis ofN-(BOC-aminoethyl)-N-(1-thyminylacetyl)-L-alanine methyl ester

To a solution of BOC-aminoethyl-(L)-alanine methyl ester (1.23 g, 5mmol) in DMF (10 mL) was added Dhbt-OH (0.9 g, 5.52 mmol) and1-thyminylacetic acid (1.01 g, 5.48 mmol). When 1-thyminylacetic aciddissolved, dichloromethane (10 mL) was added and the solution was cooledin an ice bath. After the reaction mixture had reached 0° C., DCC (1.24g, 6.01 mmol) was added. Within 5 minutes after the addition, aprecipitate of DCU was seen. After a further 5 minutes, the ice bath wasremoved. Two hours later, tlc analysis showed the reaction to becomplete. The mixture was filtered and the precipitate washed withdichloromethane (100 mL). The resulting solution was extracted twicewith 5% sodium hydrogen carbonate (150 mL) and twice with saturatedpotassium hydrogen sulfate (25 mL) in water (100 mL). After a finalextraction with saturated sodium chloride (150 mL), the solution wasdried with magnesium sulfate and evaporated to give a white foam. Thefoam was purified by column chromatography on silica gel usingdichloromethane with a methanol gradient as eluent. This yielded a purecompound (>99% by HPLC) (1.08 g, 52.4%). FAB-MS: 413 (M+1) and 431(M+1+water). ¹H-NMR (CDCl₃) δ: 4.52 (s, 2H, CH′₂); 3.73 (s, 3H, OMe);3.2-3.6 (m, 4H, ethyl CH₂′s); 1.90 (s, 3H, Me in T); 1.49 (d, 3H, Me inAla, J=7.3 Hz); 1.44 (s, 9H, BOC).

EXAMPLE 37 Synthesis ofN-(BOC-aminoethyl)-N-(1-thyminylacetyl)-L-alanine

The methyl ester of the title compound (2.07 g, 5.02 mmol) was dissolvedin methanol (100 mL), and cooled in an ice bath. 2 M Sodium hydroxide(100 mL) was added. After stirring for 10 minutes, the pH of the mixturewas adjusted to 3 with 4 M hydrogen chloride. The solution wassubsequently extracted with ethyl acetate (3×100 mL). The combinedorganic extracts were dried over magnesium sulfate. After evaporation,the resulting foam was dissolved in ethyl acetate (400 mL) and a 5 mL ofmethanol to dissolve the solid material. Petroleum ether then was addeduntil precipitation started. After allowing the mixture to standovernight at −20° C., the precipitate was removed by filtration. Thisyielded 1.01 g (50.5%) of pure compound (>99% by HPLC). The compound wasrecrystallized from 2-propanol. FAB-MS: 399 (M+1). ¹H-NMR (DMSO-d₆) δ:11.35 (s, 1H, COO); 7.42 (s, 1H, H′₆); 4.69 (s, 2H, CH′₂); 1.83 (s, 3H,Me in T); 1.50-1.40 (m, 12H, Me in Ala+BOC).

EXAMPLE 38 (a) Synthesis ofN-(BOC-aminoethyl)-N-(1-thyminylacetyl)-D-alanine methyl ester

To a solution of BOC-aminoethyl alanine methyl ester (2.48 g, 10.1 mmol)in DMF (20 mL) was added Dhbt-OH (1.8 g, 11 mmol) and thyminylaceticacid (2.14 g, 11.6 mmol). After dissolution of 1-thyminylacetic acid,methylene chloride (20 mL) was added and the solution cooled in an icebath. When the reaction mixture had reached a temperature of 0° C., DCC(2.88 g, 14 mmol) was added. Within 5 minutes of the addition, aprecipitate of DCU was seen. After 35 minutes the ice bath was removed.The reaction mixture was filtered 3.5 h later and the precipitate washedwith methylene chloride (200 mL). The resulting solution was extractedtwice with 5% sodium hydrogen carbonate (200 mL) and twice withsaturated potassium hydrogen sulfate in water (100 mL). After finalextraction with saturated sodium chloride (250 mL), the solution wasdried with magnesium sulfate and evaporated to give an oil. The oil waspurified by short column silica gel chromatography using methylenechloride with a methanol gradient as eluent. This yielded a compoundwhich was 96% pure according to HPLC (1.05 g, 25.3%) after precipitationwith petroleum ether. FAB-MS: 413 (M+1). ¹H-NMR (CDCl₃) δ: 5.64 (t, 1H,BOC—NH, J=5.89 Hz); 4.56 (d, 2H, CH′₂); 4.35 (q, 1H, CH in Ala, J=7.25Hz); 3.74 (s, 3H, OMe); 3.64-3.27 (m, 4H, ethyl H's); 1.90 (s, 3H, Me inT); 1.52-1.44 (t, 12H, BOC+Me in Ala).

(b) Synthesis of N-(BOC-aminoethyl)-N-(1-thyminylacetyl)-D-alanine

The methyl ester of the title compound (1.57 g, 3.81 mmol) was dissolvedin methanol (100 mL) and cooled in an ice bath. Sodium hydroxide (2 M,100 mL) was added. After stirring for 10 minutes, the pH of the mixturewas adjusted to 3 with 4 M hydrogen chloride. The solution was thenextracted with ethyl acetate (3×100 mL). The combined organic extractswere dried over magnesium sulfate. After evaporation, the oil wasdissolved in ethyl acetate (200 mL). Petroleum ether was added (to atotal volume of 600 mL) until precipitation started. After standingovernight at −20° C., the precipitate was removed by filtration. Thisafforded 1.02 g (67.3%) of the title compound, which was 94% pureaccording to HPLC. FAB-MS: 399 (M+1). ¹H-NMR, δ: 11.34 (s, 1H, COOH);7.42 (s, 1H, H′₆); 4.69 (s, 2H, CH′₂); 4.40 (q, 1H, CH in Ala, J=7.20Hz); 1.83 (s, 3H, Me in T); 1.52-1.40 (m, 12H, BOC+Me in Ala).

EXAMPLE 39 Synthesis ofN-(N′-BOC-3′-aminopropyl)-N-[(1-thyminyl)-acetyl]glycine methyl ester

N-(N′-BOC-3′-aminopropyl)glycine methyl ester (2.84 g, 0.0115 mol) wasdissolved in DMF (35 mL), followed by addition of DhbtOH (2.07 g, 0.0127mol) and 1-thyminylacetic acid (2.34 g, 0.0127 mol). Methylene chloride(35 mL) was added and the mixture cooled to 0° C. in an ice bath. Afteraddition of DCC (2.85 g, 0.0138 mol), the mixture was stirred at 0° C.for 2 h, followed by 1 h at room temperature. The precipitated DCU wasremoved by filtration, washed with methylene chloride (25 mL), and afurther amount of methylene chloride (150 mL) was added to the filtrate.The organic phase was extracted with sodium hydrogen carbonate (1 volumesaturated diluted with 1 volume water, 6×250 mL), potassium sulfate (1volume saturated diluted with 4 volumes water, 3×250 mL), and saturatedaqueous sodium chloride (1×250 mL), dried over magnesium sulfate, andevaporated to dryness in vacuo. The solid residue was suspended inmethylene chloride (35 mL) and stirred for 1 h. The precipitated DCU wasremoved by filtration and washed with methylene chloride (25 mL). Thefiltrate was evaporated to dryness in vacuo, and the residue purified bycolumn chromatography on silica gel, eluting with a mixture of methanoland methylene chloride (gradient from 3-7% methanol in methylenechloride). This afforded the title compound as a white solid (3.05 g,64%). M.p. 76-79° C. (decomp.). Anal. for C₁₈H₂₈N₄O₇, found (calc.) C,52.03 (52.42); H, 6.90 (6.84); N, 13.21 (13.58). The compound showedsatisfactory ¹H and ¹³C-NMR spectra.

EXAMPLE 40 Synthesis ofN-(N′-BOC-3′-aminopropyl)-N-[(1-thyminyl)-acetyl]glycine

N-(N′-BOC-3′-aminopropyl)-N-[(1-thyminyl)acetyl]glycine methyl ester(3.02 g, 0.00732 mol) was dissolved in methanol (25 mL) and stirred for1.5 h with 2 M sodium hydroxide (25 mL). Methanol was removed byevaporation in vacuo, and the pH adjusted to 2 with 4 M hydrochloricacid at 0° C. The product was isolated as white crystals by filtration,washed with water (3×10 mL), and dried over sicapent, in vacuo. Yield:2.19 g (75%). Anal. for C₁₇H₂₆N₄O₇, H₂O, found (calc.) C, 49.95 (49.03);H, 6.47 (6.29); N, 13.43 (13.45). The compound showed satisfactory ¹Hand ¹³C-NMR spectra.

EXAMPLE 41 Synthesis of 3-(1-thyminyl)propanoic acid methyl ester

Thymine (14 g, 0.11 mol) was suspended in methanol. Methyl acrylate(39.6 mL, 0.44 mol) was added, along with catalytic amounts of sodiumhydroxide. The solution was refluxed in the dark for 45 h, evaporated todryness in vacuo, and the residue dissolved in methanol (8 mL) withheating. After cooling in an ice bath, the product was precipitated byaddition of ether (20 mL), isolated by filtration, washed with ether(3×15 mL), and dried over sicapent, in vacuo. Yield: 11.23 g (48%). M.p.112-119° C. Anal. for C₉H₁₂N₂O₄, found (calc.) C, 51.14 (50.94); H, 5.78(5.70); N, 11.52 (13.20). The compound showed satisfactory ¹H and¹³C-NMR spectra.

EXAMPLE 42 Synthesis of 3-(1-thyminyl)propanoic acid

3-(1-Thyminyl)propanoic acid methyl ester (1 g, 0.0047 mol) wassuspended in 2 M sodium hydroxide (15 mL), refluxed for 10 minutes. ThepH was adjusted to 0.3 with conc. hydrochloric acid. The solution wasextracted with ethyl acetate (10×25 mL). The organic phase was extractedwith saturated aqueous sodium chloride, dried over magnesium sulfate,and evaporated to dryness in vacuo, to give the title compound as awhite solid (0.66 g, 71%). M.p. 118-121° C. Anal. for C₈H₁₀N₂O₄, found(calc.) C, 48.38 (48.49); H, 5.09 (5.09); N, 13.93 (14.14). The compoundshowed satisfactory ¹H and ¹³C-NMR spectra.

EXAMPLE 43 Synthesis ofN-(N′-BOC-aminoethyl)-N-[(1-thyminyl)-propanoyl]glycine ethyl ester

N-(N′-BOC-aminoethyl)glycine ethyl ester (1 g, 0.0041 mol) was dissolvedin DMF (12 mL). DhbtOH (0.73 g, 0.0045 mol) and 3-(1-thyminyl)propanoicacid (0.89 g, 0.0045 mol) were added. Methylene chloride (12 mL) wasthen added and the mixture was cooled to 0° C. in an ice bath. Afteraddition of DCC (1.01 g, 0.0049 mol), the mixture was stirred at 0° C.for 2 h, followed by 1 h at room temperature. The precipitated DCU wasremoved by filtration, washed with methylene chloride (25 mL), and afurther amount of methylene chloride (50 mL) was added to the filtrate.The organic phase was extracted with sodium hydrogen carbonate (1 volumesaturated diluted with 1 volume water, 6×100 mL), potassium sulfate (1volume saturated diluted with 4 volumes water, 3×100 mL), and saturatedaqueous sodium chloride (1×100 mL), dried over magnesium sulfate, andevaporated to dryness, in vacuo. The solid residue was suspended inmethylene chloride (15 mL), and stirred for 1 h. The precipitated DCUwas removed by filtration and washed with methylene chloride. Thefiltrate was evaporated to dryness in vacuo, and the residue purified bycolumn chromatography on silica gel, eluting with a mixture of methanoland methylene chloride (gradient from 1 to 6% methanol in methylenechloride). This afforded the title compound as a white solid (1.02 g,59%). Anal. for C₁₉H₃₀N₄O₇, found (calc.) C, 53.15 (53.51); H, 6.90(7.09); N, 12.76 (13.13). The compound showed satisfactory ¹H and¹³C-NMR spectra.

EXAMPLE 44 Synthesis ofN-(N′-BOC-aminoethyl)-N-[(1-thyminyl)-propanoyl]glycine

N-(N′-BOC-aminoethyl)-N-[(1-thyminyl)propanoyl]glycine ethyl ester (0.83g, 0.00195 mol) was dissolved in methanol (25 mL). Sodium hydroxide (2M, 25 mL) was added. The solution was stirred for 1 h. The methanol wasremoved by evaporation in vacuo, and the pH adjusted to 2 with 4 Mhydrochloric acid at 0° C. The product was isolated by filtration,washed with ether (3×15 mL), and dried over sicapent, in vacuo. Yield:0.769 g, 99%). M.p. 213° C. (decomp.).

EXAMPLE 45 Synthesis of Mono-BOC-ethylenediamine (2)

tert-Butyl-4-nitrophenyl carbonate (1, 10 g, 0.0418 mol) dissolved inDMF (50 mL) was added dropwise over a period of 30 minutes to a solutionof ethylenediamine (27.9 mL, 0.418 mol) and DMF (50 mL) and stirredovernight. The mixture was evaporated to dryness in vacuo, and theresulting oil dissolved in water (250 mL). After cooling to 0° C., thepH was adjusted to 3.5 with 4 M hydrochloric acid. The solution was thenfiltered and extracted with chloroform (3×250 mL). The pH was adjustedto 12 (at 0° C.) with 2 M sodium hydroxide, and the aqueous solutionextracted with methylene chloride (3×300 mL). After treatment with asolution of saturated aqueous sodium chloride (250 mL), the methylenechloride solution was dried over magnesium sulfate. After filtration,the solution was evaporated to dryness in vacuo, resulting in 4.22 g(63%) of the product as an oil. ¹H-NMR (90 MHz, CDCl₃) δ: 1.44 (s, 9H);2.87 (t, 2H); 3.1 (q, 2H); 5.62 (sb).

EXAMPLE 46 Synthesis of (N-BOC-aminoethyl)-β-alanine methyl ester.HCl

Mono-BOC-ethylenediamine (2) (16.28 g, 0.102 mol) was dissolved inacetonitrile (400 mL) and methyl acrylate (91.5 mL, 1.02 mol) wastransferred to the mixture with acetonitrile (200 mL). The solution wasrefluxed overnight under nitrogen in the dark to avoid polymerization ofmethyl acrylate. After evaporation to dryness in vacuo, a mixture ofwater and ether (200 mL+200 mL) was added, and the solution was filteredand vigorously stirred. The aqueous phase was extracted one additionaltime with ether and then freeze dried to yield a yellow solid.Recrystallization from ethyl acetate yielded 13.09 g (46%) of the titlecompound. M.p. 138-140° C. Anal. for C₁₁H₂₃N₂O₄Cl, found (calc.) C,46.49 (46.72); H, 8.38 (8.20); N, 9.83 (9.91); Cl: 12.45 (12.54). ¹H-NMR(90 MHz; DMSO-d₆) δ: 1.39 (s, 9H); 2.9 (m, 8H); 3.64 (s, 3H).

EXAMPLE 47 Synthesis ofN-[(1-Thyminyl)acetyl]-N′-BOC-aminoethyl-β-alanine methyl ester

(N-BOC-aminoethyl)-β-alanine methyl ester.HCl (3) (2 g, 0.0071 mol) and1-thyminylacetic acid pentafluorophenyl ester (5) (2.828 g, 0.00812 mol)were dissolved in DMF (50 mL). Triethyl amine (1.12 mL, 0.00812 mol) wasadded and the mixture stirred overnight. After addition of methylenechloride (200 mL), the organic phase was extracted with aqueous sodiumhydrogen carbonate (3×250 mL), half-saturated solution of aqueouspotassium hydrogen sulfate (3×250 mL), and saturated solution of aqueoussodium chloride (250 mL) and dried over magnesium sulfate. Filtrationand evaporation to dryness in vacuo, resulted in a yield of 2.9 g (99%)of product (oil). ¹H-NMR (250 MHz; CDCl₃; due to limited rotation aroundthe secondary amide several of the signals were doubled) δ: 1.43 (s,9H); 1.88 (s, 3H); 2.63 (t, 1H); 2.74 (t, 1H); 3.25-3.55 (4×t, 8H); 3.65(2×t, 2H); 3.66 (s, 1.5); 3.72 (s, 1.5); 4.61 (s, 1H); 4.72 (s, 2H);5.59 (s, 0.5H); 5.96 (s, 0.5H); 7.11 (s, 1H); 10.33 (s, 1H).

EXAMPLE 48 Synthesis ofN-[(1-thyminyl)acetyl]-N′-BOC-aminoethyl-β-alanine

N-[(1-Thyminyl)acetyl]-N′-BOC-aminoethyl-β-alanine methyl ester (3 g,0.0073 mol) was dissolved in 2 M sodium hydroxide (30 mL), the pHadjusted to 2 at 0° C. with 4 M hydrochloric acid, and the solutionstirred for 2 h. The precipitate was isolated by filtration, washedthree times with cold water, and dried over sicapent, in vacuo. Yield:2.23 g (77%). M.p. 170-176° C. Anal. for C₁₇H₂₆N₄O₇, H₂O, found (calc.)C, 49.49 (49.03) H, 6.31 (6.78) N, 13.84 (13.45). ¹H-NMR (90 MHz;DMSO-d₆) δ: 1.38 (s, 9H); 1.76 (s, 3H); 2.44 and 3.29 (m, 8H); 4.55 (s,2H); 7.3 (s, 1H); 11.23 (s, 1H). FAB-MS: 399 (M+1).

EXAMPLE 49 Synthesis ofN-[(1-(N⁴-Z)-cytosinyl)acetyl]-N′-BOC-aminoethyl-β-alanine methyl ester

(N-BOC-amino-ethyl)-β-alanine methyl ester.HCl (3) (2 g, 0.0071 mol) and1-(N-4-Z)-cytosinylacetic acid pentafluorophenyl ester (5) (3.319 g,0.0071 mol) were dissolved in DMF (50 mL). Triethyl amine (0.99 mL,0.0071 mol) was added and the mixture stirred overnight. After additionof methylene chloride (200 mL), the organic phase was extracted withaqueous sodium hydrogen carbonate (3×250 mL), half-saturated solution ofaqueous potassium hydrogen sulfate (3×250 mL), and saturated solution ofaqueous sodium chloride (250 ml), and dried over magnesium sulfate.Filtration and evaporation to dryness, in vacuo, resulted in 3.36 g ofsolid compound which was recrystallized from methanol. Yield: 2.42 g(64%). M.p. 158-161° C. Anal. for C₂₅H₃₃N₅O₈, found (calc.) C, 55.19(56.49); H, 6.19 (6.26); N, 12.86 (13.18). ¹H-NMR (250 MHz; CDCl₃; dueto limited rotation around the secondary amide several of the signalswere doubled) δ: 1.43 (s, 9H); 2.57 (t, 1H); 3.60-3.23 (m's, 6H); 3.60(s, 5H); 3.66 (s, 1.5H); 4.80 (s, 1H); 4.88 (s, 1H); 5.20 (s, 2H);7.80-7.25 (m's, 7H). FAB-MS: 532 (M+1).

EXAMPLE 50 Synthesis ofN-[(1-(N⁴-Z)-cytosinyl)acetyl]-N′-BOC-aminoethyl-β-alanine

N-[(1-(N4-Z)-cytosinyl)acetyl]-N′-BOC-aminoethyl-β-alanine methyl ester(0.621 g, 0.0012 mol) was dissolved in 2 M sodium hydroxide (8.5 mL) andstirred for 2 h. Subsequently, the pH was adjusted to 2 at 0° C. with 4M hydrochloric acid and the solution stirred for 2 h. The precipitatewas isolated by filtration, washed three times with cold water, anddried over sicapent, in vacuo. Yield: 0.326 g (54%). The white solid wasrecrystallized from 2-propanol and washed with petroleum ether. Mp. 163°C. (decomp.). Anal. for C₂₄H₃₁N₅O₈, found (calc.) C, 49.49 (49.03); H,6.31 (6.78); N, 13.84 (13.45). ¹H-NMR (250 MHz; CDCl₃, due to limitedrotation around the secondary amide several of the signals were doubled)δ: 1.40 (s, 9H); 2.57 (t, 1H); 2.65 (t, 1H); 3.60-3.32 (m's, 6H); 4.85(s 1H); 4.98 (s, 1H); 5.21 (s, 2H); 5.71 (s, 1H, broad); 7.99-7.25 (m's,7H). FAB-MS: 518 (M+1).

EXAMPLE 51

Solid Phase Synthesis of H-[Taeg]₅-[Gaeg]-[Taeg]₄-Lys-NH₂

The protected PNA was assembled onto a BOC-Lys(ClZ) modified MBHA resinwith a substitution of approximately 0.15 mmol/g (determined byquantitative Ninhydrin reaction). Capping of only uncoupled amino groupswas carried out before the incorporation of the BOC-Gaeg-OH monomer.

Stepwise Assembly of H-[Taeg]₅-[Gaeg]-[Taeg]₄-Lys-NH₂ (SyntheticProtocol)

Synthesis was initiated on 102 mg (dry weight) of preswollen (overnightin DCM) and neutralized BOC-Lys(ClZ)-MBHA resin. The steps performedwere as follows: (1) BOC-deprotection with TFA/DCM (dichloromethane)(1:1, v/v), 1×2 minutes and 1×0.5 h, 3 mL; (2) washing with DCM, 4×20seconds, 3 mL; washing with DMF, 2×20 seconds, 3 mL; washing with DCM,2×20 seconds, 3 mL, and drain for 30 seconds; (3) neutralization withDIEA/DCM (1:19 v/v), 2×3 minutes, 3 mL; (4) washing with DCM, 4×20seconds, 3 mL, and drain for 1 minute; (5) addition of 4 equivalents ofdiisopropyl carbodiimide (0.06 mmol, 9.7 μL) and 4 equivalents ofBOC-Taeg-OH (0.06 mmol, 24 mg) or BocGaeg-OH (0.06 mmol, 30 mg)dissolved in 0.6 mL of 1:1 (v/v) DCM/DMF (final concentration of monomer0.1 M), the coupling reaction was allowed to proceed for 0.5 h whileshaking at room temperature; (6) drain for 20 seconds; (7) washing withDMF, 2×20 seconds and 1×2 minutes, 3 mL; washing with DCM 4×20 seconds,3 mL; (8) neutralization with DEEA/DCM (1:19 v/v), 2×3 minutes, 3 mL;(9) washing with DCM 4×20 seconds, 3 mL, and drain for 1 minute; (10)qualitative Kaiser test; (11) blocking of unreacted amino groups byacetylation with Ac₂O/pyridine/DCM (1:1:2, v/v), 1×0.5 h, 3 mL; and (12)washing with DCM, 4×20 seconds, 2×2 minutes and 2×20 seconds, 3 mL.Steps 1-12 were repeated until the desired sequence was obtained. Allqualitative Kaiser tests were negative (straw-yellow colour with nocoloration of the beads) indicating near 100% coupling yield. The PNAoligomer was cleaved and purified by the normal procedure. FAB-MS:2832.11 [M+1] (calc. 2832.15)

EXAMPLE 52

Solid Phase Synthesis of H-Taeg-Aaeg-[Taeg]₈-Lys-NH₂

(a) Stepwise Assembly of BOC-Taeg-A(Z)aeg-[Taeg]₈-Lys(ClZ)-MBHA Resin

About 0.3 g of wet BOC-[Taeg]₈-Lys(ClZ)-MBHA resin was placed in a 3 mLSPPS reaction vessel. BOC-Taeg-A(Z)aeg-[Taeg]₈-Lys(ClZ)-MBHA resin wasassembled by in situ DCC coupling (single) of the A(Z)aeg residueutilizing 0.19 M of BOC-A(Z)aeg-OH together with 0.15 M DCC in 2.5 mL of50% DMF/CH₂Cl₂ and a single coupling with 0.15 M BOC-Taeg-OPfp in neatCH₂Cl₂ (“Synthetic Protocol 5”). The synthesis was monitored by thequantitative ninhydrin reaction, which showed about 50% incorporation ofA(Z)aeg and about 96% incorporation of Taeg.

(b) Cleavage, Purification, and Identification ofH-Taeg-Aaeg-[Taeg]₈-Lys-NH₂

The protected BOC-Taeg-A(Z)aeg-[Taeg]₈-Lys(ClZ)-BHA resin was treated asdescribed in Example 17(c) to yield about 15.6 mg of crude material uponHF cleavage of 53.1 mg dry H-Taeg-A(Z)aeg-[Taeg]₈-Lys(ClZ)-BHA resin.The main peak at 14.4 minutes accounted for less than 50% of the totalabsorbance. A 0.5 mg portion of the crude product was purified to giveapproximately 0.1 mg of H-Taeg-Aaeg-[Taeg]₈-Lys-NH₂. For (MH+)⁺ thecalculated m/z value was 2816.16 and the measured m/z value was 2816.28.

(c) Synthetic Protocol 5

(1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 2.5 mL, 3×1 minute and1×30 minutes; (2) washing with CH₂Cl₂, 2.5 mL, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2.5 mL, 3×2 minutes; (4)washing with CH₂Cl₂, 2.5 mL, 6×1 minute, and drain for 1 minute; (5) 2-5mg sample of PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the substitution; (6)addition of 0.47 mmol (0.25 g) BOC-A(Z)aeg-OH dissolved in 1.25 mL ofDMF followed by addition of 0.47 mmol (0.1 g) DCC in 1.25 mL of CH₂Cl₂or 0.36 mmol (0.2 g) BOC-Taeg-OPfp in 2.5 mL of CH₂Cl₂; the couplingreaction was allowed to proceed for a total of 20-24 h while shaking;(7) washing with DMF, 2.5 mL, 1×2 minutes; (8) washing with CH₂Cl₂, 2.5mL, 4×1 minute; (9) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2.5 mL,2×2 minutes; (10) washing with CH₂Cl₂, 2.5 mL, 6×1 minute; (11) 2-5 mgsample of protected PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the extent of coupling;(12) blocking of unreacted amino groups by acetylation with a 25 mLmixture of acetic anhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h(except after the last cycle); and (13) washing with CH₂Cl₂, 2.5 mL, 6×1minute; (14) 2×2-5 mg samples of protected PNA-resin are removed,neutralized with DIEA/CH₂Cl₂ (1:19, v/v) and washed with CH₂Cl₂ forninhydrin analyses.

EXAMPLE 53

Solid Phase Synthesis of H-[Taeg]₂-Aaeg-[Taeg]₅-Lys-NH₂

(a) Stepwise Assembly of BOC-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(ClZ)-MBHA Resin

About 0.5 g of wet BOC-[Taeg]₅-Lys(ClZ)-MBHA resin was placed in a 5 mLSPPS reaction vessel. BOC-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(ClZ)-MBHA resinwas assembled by in situ DCC coupling of both the A(Z)aeg and the Taegresidues utilising 0.15 M to 0.2 M of protected PNA monomer (free acid)together with an equivalent amount of DCC in 2 mL neat CH₂Cl₂(“Synthetic Protocol 6”). The synthesis was monitored by thequantitative ninhydrin reaction which showed a total of about 82%incorporation of A(Z)aeg after coupling three times (the first couplinggave about 50% incorporation; a fourth HOBt-mediated coupling in 50%DMF/CH2Cl2 did not increase the total coupling yield significantly) andquantitative incorporation (single couplings) of the Taeg residues.

(b) Cleavage, Purification, and Identification ofH-[Taeg]₂-Aaeg-[Taeg]₅-Lys-NH₂

The protected BOC-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(ClZ)-BHA resin was treatedas described in Example 17(c) to yield about 16.2 mg of crude materialupon HF cleavage of 102.5 mg dry H-[Taeg]₂-A(Z)aeg-[Taeg]₅-Lys(ClZ)-BHAresin. A small portion of the crude product was purified. For (MH+)⁺,the calculated m/z value was 2050.85 and the measured m/z value was2050.90

(c) Synthetic Protocol 6

(1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 2 mL, 3×1 minute and1×30 minutes; (2) washing with CH₂Cl₂, 2 mL, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2 mL, 3×2 minutes; (4)washing with CH₂Cl₂, 2 mL, 6×1 minute, and drain for 1 minute; (5) 2-5mg sample of PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the substitution; (6)addition of 0.44 mmol (0.23 g) BOC-A(Z)aeg-OH dissolved in 1.5 mL ofCH₂Cl₂ followed by addition of 0.44 mmol (0.09 g) DCC in 0.5 mL ofCH₂Cl₂ or 0.33 mmol (0.13 g) BOC-Taeg-OH in 1.5 mL of CH₂Cl₂ followed byaddition of 0.33 mmol (0.07 g) DCC in 0.5 mL of CH₂Cl₂; the couplingreaction was allowed to proceed for a total of 20-24 h with shaking; (7)washing with DMF, 2 mL, 1×2 minutes; (8) washing with CH₂Cl₂, 2 mL, 4×1minute; (9) neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 2 mL, 2×2minutes; (10) washing with CH₂Cl₂, 2 mL, 6×1 minute; (11) 2-5 mg sampleof protected PNA-resin was removed and dried thoroughly for aquantitative ninhydrin analysis to determine the extent of coupling;(12) blocking of unreacted amino groups by acetylation with a 25 mLmixture of acetic anhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h(except after the last cycle); (13) washing with CH₂Cl₂, 2 mL, 6×1minute; and (14) 2×2-5 mg samples protected PNA-resin were removed,neutralized with DIEA/CH₂Cl₂ (1:19, v/v) and washed with CH₂Cl₂ forninhydrin analyses.

EXAMPLE 54

Hybridization Experiments

The PNA oligomer H-T₄C₂TCT-LysNH₂ (SEQ. ID NO: 48) was preparedaccording to the procedure described in Example 51. Hybridizationexperiments with this sequence should resolve the issue of orientation,since it is truly asymmetrical. Such experiments should also resolve theissues of pH-dependency of the T_(m), and the stoichiometry of complexesformed.

Hybridization experiments with the PNA oligomer H-T₄C₂TCTC-LysNH₂ wereperformed as follows:

T_(m) Row Hybridized With pH (° C.) § 1 5'-(dA)₄(dG)₂(dA)(dG)(dA)(dG)(SEQ. ID NO.: 49) 7.2 55.5 2:1 2 5'-(dA)₄(dG)₂(dA)(dG)(dA)(dG) (SEQ. IDNO.: 49) 9.0 26.0 2:1 3 5'-(dA)₄(dG)₂(dA)(dG)(dA)(dG) (SEQ. ID NO.: 49)5.0 88.5 2:1 4 5'-(dG)(dA)(dG)(dA)(dG)₂(dA)₄ (SEQ. ID NO.: 50) 7.2 38.02:1 5 5'-(dG)(dA)(dG)(dA)(dG)₂(dA)₄ (SEQ. ID NO.: 50) 9.0 31.5 — 65'-(dG)(dA)(dG)(dA)(dG)₂(dA)₄ (SEQ. ID NO.: 50) 5.0 52.5 — 75'-(dA)₄(dG)(dT)(dA)(dG)(dA)(dG) (SEQ. ID NO.: 51) 7.2 39.0 — 85'-(dA)₄(dG)(dT)(dA)(dG)(dA)(dG) (SEQ. ID NO.: 51) 9.0 <20 — 95'-(dA)₄(dG)(dT)(dA)(dG)(dA)(dG) (SEQ. ID NO.: 51) 5.0 51.5 — 105'-(dA)₄(dG)₂(dT)(dG)(dA)(dG) (SEQ. ID NO.: 52) 7.2 31.5 — 115'-(dA)₄(dG)₂(dT)(dG)(dA)(dG) (SEQ. ID NO.: 52) 5.0 50.5 — 125'-(dG)(dA)(dG)(dA)(dT)(dG)(dA)₄ (SEQ. ID NO.: 53) 7.2 24.5 — 135'-(dG)(dA)(dG)(dA)(dT)(dG)(dA)₄ (SEQ. ID NO.: 53) 9.0 <20 — 145'-(dG)(dA)(dG)(dA)(dT)(dG)(dA)₄ (SEQ. ID NO.: 53) 5.0 57.0 — 155'-(dG)(dA)(dG)(dT)(dG)₂(dA)₄ (SEQ. ID NO.: 54) 7.2 25.0 — 165'-(dG)(dA)(dG)(dT)(dG)₂(dA)₄ (SEQ. ID NO.: 54) 5.0 39.5 — 52.0 § =stoichiometry determined by UV-mixing curves — = not determined

These results show that a truly mixed sequence gave rise to well-definedmelting curves. The PNA oligomers can actually bind in both orientations(compare row 1 and 4), although there is a preference for theN-terminal/5′-orientation. Introducing a single mismatch opposite eitherT or C caused a lowering of T_(m) by more than 16° C. at pH 7.2; at pH5.0 the T_(m) was lowered more than 27° C. This shows that there is avery high degree a sequence-selectivity which should be a generalfeature for all PNA C/T sequences.

As indicated above, there is a very strong pH-dependency for the T_(m),indicating that Hoogsteen basepairing is important for the formation ofhybrids. Therefore, it is not surprising that the stoichiometry wasfound to be 2:1.

The lack of symmetry in the sequence and the very large decrease inT_(m) when mismatches are present show that the Watson-Crick strand andthe Hoogsteen strand are parallel when bound to complementary DNA. Thisis true for both the orientations, i.e., 5′/N-terminal and3′/N-terminal.

EXAMPLE 55

T_(m)s of PNA Oligomers

The results of hybridization experiments with H-T₅GT₄-LysNH₂ were asfollows:

Row Deoxyoligonucleotide T_(m) (° C.) 1 5'-(dA)₅(dA)(dA)₄-3' (SEQ. IDNO. 55) 55.0 2 5'-(dA)₅(dG)(dA)₄-3' (SEQ. ID NO. 56) 47.0 35'-(dA)₅(dG)(dA)₄-3' (SEQ. ID NO. 56) 56.5 4 5'-(dA)₅(dT)(dA)₄-3' (SEQ.ID NO. 57) 46.5 5 5'-(dA)₄(dG)(dA)₅-3' (SEQ. ID NO. 58) 48.5 65'-(dA)₄(dC)(dA)₅-3' (SEQ. ID NO. 59) 55.5 7 5'-(dA)₄(dT)(dA)₅-3' (SEQ.ID NO. 60) 47.0

As observed by comparing rows 1, 3, and 6 with rows 2, 4, 5, and 7, Gcan, in this mode, discriminate between C/A and G/T in the DNA strand,i.e., sequence discrimination is observed. The complex in row 3 wasfurthermore determined to be 2 PNA: 1 DNA complex by UV-mixing curves.

EXAMPLE 56

Synthesis of PNA 15-mer Containing Four Naturally Occurring Nucleobases:H-[Taeg]-[Aaeg]-[Gaeg]-[Taeg]-[Taeg]-[Aaeg]-[Taeg]-[Caeg]-[Taeg]-[Caeg]-[Taeg]-[Aaeg]-[Taeg]-[Caeg]-[Taeg]-Lys-NH₂

The protected PNA was assembled onto a BOC-Lys(ClZ) modified MBHA resinwith a substitution of approximately 0.145 mmol/g. Capping of onlyuncoupled amino groups was carried out before the incorporation of theBOC-Gaeg-OH monomer.

Synthesis was initiated on 100 mg (dry weight) of neutralisedBOC-Lys(ClA)-MBHA resin that had been preswollen overnight in DCM. Theincorporation of the monomers followed the protocol of Example 28,except at step 5 for the incorporation of the BOC-Aaeg-OH monomer. Step5 for the present synthesis involved addition of 4 equivalents ofdiisopropyl carbodiimide (0.06 mM, 9.7 μL) and 4 equivalents ofBOC-Aaeg-OH (0.06 mmol, 32 mg) dissolved in 0.6 mL of DCM/DMF (1:1, v/v)(final concentration of monomer 0.1M). The coupling reaction was allowedto proceed for 1×15 minutes and 1×60 minutes (recoupling).

All qualitative Kaiser tests were negative (straw-yellow color with nocoloration of the beads). The PNA oligomer was cleaved and purified bythe standard procedure. FAB-MS average mass found(calc.) (M+H) 4145.1(4146.1).

EXAMPLE 57

Solid Phase Synthesis ofH-[Taeg]₂-Aaeg-Taeg-Caeg-Aaeg-Taeg-Caeg-Taeg-Caeg-Lys-NH2

(a) Stepwise Assembly ofBOC-[Taeg]₂-A(Z)aeg-Taeg-C(Z)aeg-A(Z)aeg-Taeg-C(Z)aeg-Taeg-C(Z)aeg-Lys(ClZ)-MBHA Resin

About 1 g of wet BOC-Lys(ClZ)-MBHA (0.28 mmol Lys/g) resin was placed ina 5 mL SPPS reaction vessel.BOC-[Taeg]2-A(Z)aeg-Taeg-C(Z)aeg-A(Z)aeg-Taeg-C(Z)aeg-Taeg-C(Z)aeg-Lys(ClZ)-MBHAresin was assembled by in situ DCC coupling of the five first residuesutilizing 0.16 M of BOC—C[Z]—OH, BOC-Taeg-OH or BOC—A(Z)aeg-OH, togetherwith 0.16 M DCC in 2 mL of 50% DMF/CH₂Cl₂ and by analogous in situ DICcoupling of the five last residues. Each coupling reaction was allowedto proceed for a total of 20-24 h with shaking. The synthesis wasmonitored by the ninhydrin reaction, which showed nearly quantitativeincorporation of all residues except of the first A(Z)aeg residue, whichhad to be coupled twice. The total coupling yield was about 96% (firstcoupling, about 89% efficiency).

(b) Cleavage, Purification, and Identification ofH-[Taeg]2-Aaeg-Taeg-Caeg-Aaeg-Taeg-Caeg-Taeg-Caeg-Lys-NH₂

The protectedBOC-[Taeg]2-A(Z)aeg-Taeg-C(Z)aeg-A(Z)aeg-Taeg-C(Z)aeg-Taeg-C(Z)aeg-Lys(ClZ)-MBHAresin was treated as described in Example 17(c) to yield about 53.4 mgof crude material upon HF cleavage of 166.1 mg of dryBOC-[Taeg]2-A(Z)aeg-Taeg-C(Z)aeg-A(Z)aeg-Taeg-C(Z)aeg-Taeg-C(Z)aeg-Lys(ClZ)-MBHAresin. The crude product (53.4 mg) was purified to give 18.3 mg ofH-[Taeg]2-Aaeg-Taeg-Caeg-Aaeg-Taeg-Caeg-Taeg-Caeg-Lys-NH₂. For (M+H)+,the calculated m/z value=2780.17 and the measured m/z value=2780.07.

EXAMPLE 58

Solid Phase Synthesis of H-[Taeg]₅-Lys(CIZ)-MBHA Resin

The PNA oligomer was assembled on 500 mg (dry weight) of MHA resin thathad been preswollen overnight in DCM. The resin was initiallysubstituted with approximately 0.15 mmol/g BOC-Lys(ClZ) as determined byquantitative ninhydrin reaction. The stepwise synthesis of the oligomerfollowed the synthetic protocol described in Example 28 employing 0.077g (0.2 mmnol) BOC-Taeg-OH and 31.3 μL (0.2 mmol) ofdiisopropylcarbodiimide in 2 mL of 50% DMF/CH₂Cl₂ in each coupling.Capping of uncoupled amino groups was carried out before deprotection ineach step. All qualitative Kaiser tests were negative indicating near100% coupling yield.

EXAMPLE 59

Synthesis of the Backbone Moiety for Scale-up By Reductive Amination

(a) Preparation of BOC-aminoacetaldehyde

3-Amino-1,2-propanediol (80 g, 0.88 mol) was dissolved in water (1500mL) and the solution was cooled to 4° C., after which BOC-anhydride (230g, 1.05 mol) was added in one portion. The solution was gently heated toroom temperature in a water bath. The pH was maintained at 10.5 by thedropwise addition of sodium hydroxide. Over the course of the reaction,a total of 70.2 g of NaOH, dissolved in 480 mL of water, was added.After stirring overnight, ethyl acetate (1000 mL) was added, the mixturecooled to 0° C. and the pH adjusted to 2.5 by the addition of 4 Mhydrochloric acid. The ethyl acetate layer was removed and the acidicaqueous solution was extracted with more ethyl acetate (8×500 mL). Thecombined ethyl acetate solution was reduced to a volume of 1500 mL usinga rotary evaporator. The resulting solution was washed with halfsaturated potassium hydrogen sulphate (1500 mL) and then with saturatedsodium chloride. It then was dried over magnesium sulphate andevaporated to dryness, in vacuo. Yield: 145.3 g (86%).

3-BOC-amino-1,2-propanediol (144.7 g, 0.757 mol) was suspended in water(750 mL) and potassium periodate (191.5 g, 0.833 mol) was added. Themixture was stirred under nitrogen for 2.5 h and the precipitatedpotassium iodate was removed by filtration and washed once with water(100 mL). The aqueous phase was extracted with chloroform (6×400 mL).The chloroform extracts were dried and evaporated to dryness, in vacuo.Yield: 102 g (93%) of an oil. BOC-aminoacetaldehyde was purified bykugelrohr distillation at 84° C. and 0.3 mmHg, in two portions. Yield:79 g (77%) as a colorless oil.

(b) Preparation of (N′-BOC-aminoethyl)glycine Methyl Ester

Palladium on carbon (10%, 2.00 g) was added to a solution ofBOC-aminoacetaldehyde (10 g, 68.9 mmol) in methanol (150 mL) at 0° C.Sodium acetate (11.3 g, 138 mmol) in methanol (150 mL), and glycinemethyl ester hydrochloride (8.65 g; 68.9 mmol) in methanol (75 mL) wereadded. The mixture was hydrogenated at atmospheric pressure for 2.5 h,then filtered through celite and evaporated to dryness, in vacuo. Thematerial was redissolved in water (150 mL) and the pH adjusted to 8 with0.5 N NaOH. The aqueous solution was extracted with methylene chloride(5×150 mL). The combined extracts were dried over sodium sulphate andevaporated to dryness, in vacuo. This resulted in 14.1 g (88%) yield of(N′-BOC-aminoethyl)glycine methyl ester. The crude material was purifiedby kugelrohr destination at 120° C. and 0.5 mm Hg to give 11.3 g (70%)of a colorless oil. The product had a purity that was higher than thematerial produced in example 26 according to tlc analysis (10% methanolin methylene chloride).

Alternatively, sodium cyanoborohydride can be used as reducing agentinstead of hydrogen (with Pd(C) as catalyst), although the yield (42%)was lower.

(c) Preparation of (N′-BOC-aminoethyl)glycine ethyl ester

The title compound was prepared by the above procedure with glycineethyl ester hydrochloride substituted for glycine methyl esterhydrochloride. Also, the solvent used was ethanol. The yield was 78%.

EXAMPLE 60

Solid Phase Synthesis of H-Tyr-[Taeg]₁₀-Lys-NH₂

(a) Stepwise Assembly of BOC-Tyr(BrZ)-[Taeg]₁₀-Lys(ClZ)-MBHA Resin

About 0.2 g of wet BOC-[Taeg]₁₀-Lys(ClZ)-MBHA resin was placed in a 5 mLSPPS reaction vessel. BOC-Tyr(BrZ)-[Taeg]₁₀-Lys(ClZ)-MBHA resin wasassembled by standard in situ DCC coupling utilizing 0.32 M ofBOC-CTyr(BrZ)-OH together with 0.32 M DCC in 3 mL neat CH₂Cl₂,overnight. The ninhydrin reaction showed about 97% incorporation ofBOC-Tyr(BrZ).

(b) Cleavage, Purification, and Identification of H-Tyr-[Taeg]₁₀-Lys-NH₂

The protected BOC-Tyr(BrZ)-[Taeg]₁₀-Lys(ClZ)-MBHA resin was treated asdescribed in Example 17(c) to yield about 5.5 mg of crude material uponHF cleavage of 20.7 mg of dry H-Tyr(BrZ)-[Taeg]₁₀-Lys(ClZ)-MBHA resin.The crude product was purified to give 2.5 mg of H-Tyr-[Taeg]₁₀-Lys-NH₂.

EXAMPLE 61

Solid Phase Synthesis of Dansyl-[Taeg]₁₀-Lys-NH₂

(a) Stepwise Assembly of Dansyl-[Taeg]₁₀-Lys(ClZ)-MBRA Resin

About 0.3 g of wet BOC-[Taeg]₁₀-Lys(ClZ)-MBHA resin was placed in a 5 mLSPPS reaction vessel. Dansyl-[Taeg]₁₀-Lys(ClZ)-MBHA resin was assembledby coupling of 0.5 M dansyl-Cl in 2 mL of pyridine, overnight. Theninhydrin reaction showed about 95% incorporation of the dansyl group.

(b) Cleavage, Purification, and Identification ofDansyl-[Taeg]₁₀-Lys-NH₂

The protected dansyl-[Taeg]₁₀-Lys(ClZ)-MBHA resin was treated asdescribed in Example 17(c) to yield about 12 mg of crude material uponHF cleavage of 71.3 mg of dry dansyl-[Taeg]₁₀-Lys(ClZ)-MBHA resin. Thecrude product was purified to give 5.4 mg of dansyl-[Taeg]₁₀-Lys-NH₂.

EXAMPLE 62

Solid Phase Synthesis of H-[Taeg]₃-Caeg-[Taeg]₄-NH₂

(a) Stepwise Assembly of BOC-[Taeg]₃-C(Z)aeg-[Taeg]₄-MBHA Resin

About 0.2 g of the above-mentioned MBHA resin was placed in a 5 mL SPPSreaction vessel and neutralized. BOC-[Taeg]₃-C(Z)aeg-[Taeg]₄-MBHA resinwas assembled by single in situ DCC coupling of the C(Z)aeg residueutilizing 0.13 M of BOC—C[Z]aeg-OH together with 0.13 M DCC in 2.5 mL of50% DMF/CH₂Cl₂ and by coupling the Taeg residues with 0.13 MBOC-Taeg-OPfp in 2.5 mL of CH₂Cl₂. Each coupling reaction was allowed toproceed with shaking overnight. The synthesis was monitored by theninhydrin reaction, which showed close to quantitative incorporation ofall the residues.

(b) Cleavage, Purification, and Identification ofH-[Taeg]₃-Caeg-[Taeg]₄NH₂

The protected BOC-[Taeg]₃-C(Z)aeg-[Taeg]₄-MBHA resin was treated asdescribed in Example 17(c) to yield about 44.4 mg of crude material uponHF cleavage of about 123 mg of dry H-[Taeg]₃-C(Z)aeg-[Taeg]₄-MBHA resin.Crude product (11 mg) was purified to give 3.6 mg ofH-[Taeg]₃-Caeg-[Taeg]₄-NH₂.

EXAMPLE 63

Solid Phase Synthesis of H-[Taeg]₂-Caeg-[Taeg]₂-Caeg-[Taeg]₄-Lys-NH₂

(a) Stepwise Assembly ofBOC-[Taeg]₂-C(Z)aeg-[Taeg]₂-C(Z)aeg-[Taeg]₄-Lys(ClZ)-MBHA Resin

About 0.3 g of wet H-[Taeg]₂-C(Z)aeg-[Taeg]₄-Lys(ClZ)-MBHA resin fromthe earlier synthesis of BOC-[Taeg]₅-C(Z)aeg-[Taeg]₄-Lys(ClZ)-MBHA resinwas placed in a 5 mL SPPS reaction vessel. After coupling of the nextresidue five times, a total incorporation of BOC—C(Z)aeg of 87% wasobtained. The five repeated couplings were carried out with 0.18 MBOC—C(Z)aeg-OPfp in 2 mL of TFE/CH₂Cl₂ (1:2, v/v), 2 mL of TFE/CH₂Cl₂(1:2, v/v), 2 mL of TFE/CH₂Cl₂ (1:2, v/v) with two drops of dioxane andtwo drops of DIEA (this condition gave only a few per cent couplingyield), 2 mL of TFE/CH₂Cl₂ (1:2, v/v) plus 0.5 g phenol and 1 mL ofCH₂Cl₂ plus 0.4 g of phenol, respectively. The two final Taeg residueswere incorporated close to quantitatively by double couplings with 0.25M BOC-Taeg-OPfp in 25% phenol/CH₂Cl₂. Al couplings were allowed toproceed overnight.

(b) Cleavage, Purification, and Identification ofH-[Taeg]₂-Caeg-[Taeg]₂-Caeg-[Taeg]₄-Lys-NH₂

The protected BOC-[Taeg]₂-C(Z)aeg-[Taeg]₂-C(Z)aeg-[Taeg]₄-Lys(ClZ)-MBHAresin was treated as described in Example 17(c) to yield about 7 mg ofcrude material upon HF cleavage of 80.7 mg of dryH-[Taeg]₂-C(Z)aeg-[Taeg]₂-C(Z)aeg-[Taeg]₄-Lys(ClZ)-MBHA resin. The crudeproduct was purified to give 1.2 mg ofH-[Taeg]₂-Caeg-[Taeg]₂-Caeg-[Taeg]₄-Lys-NH₂ (>99.9% purity).

EXAMPLE 64

Alternative Protecting Group Strategy for PNA Synthesis

(a) Synthesis of Test Compounds

2-Amino-6-O-benzyl purine: To a solution of 2.5 g (0.109 mol) of sodiumin 100 mL of benzyl alcohol was added 10.75 g (0.063 mol) of2-amino-6-chloropurine. The mixture was stirred for 12 h at 120° C. Thesolution was cooled to room temperature and neutralized with acetic acidand extracted with 10 portions of 50 mL of 0.2 N sodium hydroxide. Thecollected sodium hydroxide phases were washed with 100 mL of diethylether and neutralized with acetic acid, whereby precipitation starts.The solution was cooled to 0° C. and the yellow precipitate wascollected by filtration. Recrystallization from ethanol gave 14.2 g(92%) of pure white crystals of the target compound. ¹H-NMR (250 MHz,DMSO-d₆) δ: 7.92 (8H); 7.60-7.40 (benzyl aromatic); 6.36 (2-NH₂); 5.57(benzyl CH₂).

(2-Amino-6-O-benzyl purinyl)methylethanoate: A mixture of 5 g (0.0207mol) of 2-amino-6-O-benzylpurine, 30 mL of DMF and 2.9 g (0.021 mol) ofpotassium carbonate was stirred at room temperature. Methyl bromoacetate(3.2 g, 1.9 mL, 0.0209 mol) was added dropwise. The solution wasfiltrated after 4 h and the solvent was removed under reduced pressure(4 mm Hg, 40° C.). The residue was recrystallized two times from ethylacetate to give 3.7 g (57%) of the target compound. ¹H-NMR (250 MHz,DMSO-d₆) δ: 7.93 (8H); 7.4-7.6 (benzyl aromatic); 6.61 (2-NH₂); 5.03(benzyl CH₂); 5.59 (CH₂); 3.78 (OCH₃).

(2-N-p-Toluenesulfonamido-6-O-benzylpurinyl)methyl ethanoate: To asolution of 0.5 g (1.6 mmol) of (2-amino-6-O-benzylpurinyl)methylethanoate in 25 mL of methylene chloride was added 0.53 g (1.62 mmol) ofp-toluenesulfonic anhydride and 0.22 g (1.62 mmol) of potassiumcarbonate. The mixture was stirred at room temperature. The mixture wasthen filtered and the solvent removed at reduced pressure (15 mm Hg, 40°C.). Diethyl ether was added to the oily residue. The resulting solutionwas stirred overnight, whereby the target compound (0.415 mg, 55%)precipitated and was collected by filtration. ¹H-NMR (250 MHz, DMSO-d₆)δ: 8.97 (8H); 7.2-7.8 (aromatic); 5.01 (benzyl CH₂); 4.24 (CH₂); 3.73(OCH₃); 2.43 (CH₃).

(b) Stability of the Tosyl Protected Base Residue in TFA and HF

The material was subjected to the standard deprotection conditions(TFA-deprotection) and the final cleavage conditions with HF. Theproducts were then subjected to HPLC-analysis using a 4μ RCM 8×10 Novapack column and solvents A (0.1% TFA in water) and B (0.1% TFA inacetonitrile) according to the following time gradient with a flow of 2mL/minute.

Time % A % B  0 100  0  5 100  0 35  0 100 37  0 100 39 100  0

The following retention times were observed: (a) Compound 1: 30.77minutes; (b) compound 2: 24.22 minutes; and (c) compound 3: 11.75minutes. The analysis showed that the O⁶-benzyl group was removed bothby TFA and HF, whereas there was no cleavage of the tosyl group in TFA,but quantitative removal in HF under the standard cleavage conditions.

EXAMPLE 65 Synthesis of 5-bromouracil-N¹-methyl acetate

5-Bromouracil (5 g, 26.2 mmol) and potassium carbonate (7.23 g, 52.3mmol) were suspended in DMF (75 mL). Methyl bromoacetate (2.48 mL, 26.1mmol) was added over a period of 5 minutes. The suspension was stirredfor 2 h at room temperature and then filtered. The solid residue waswashed twice with DMF, and the combined filtrates were evaporated todryness, in vacuo. The residue was an oil containing the title compound,DMF and some unidentified impurities. It was not necessary to purify thetitle compound before hydrolysis. ¹H-NMR (DMSO-d₆, 250 MHz) δ: 8.55(impurity); 8.27 (CBr═CHN); 8.02 (impurity); 4.76 (impurity); 4.70(impurity); 4.62 (NCH ₂COOCH₃); 3.78 (COOCH ₃); 2.96 (DMF); 2.80 (DMF).¹³C-NMR (DMSO-d₆, 250 MHz) ppm: 168.8 COOCH ₃); 172.5 (CH═CBrCON); 161.6(DMF); 151.9 (NCON); 145.0 (CO—CBr═CHN); 95.6 (COCBr═CHN); 52.6(impurity); 52.5 (OCH₃); 49.7 (impurity); 48.8 (NCH₂COOMe); 43.0(impurity); 36.0 (DMF). UV(Methanol; nm_(max)); 226; 278. IR (KBr;cm⁻¹_(—); 3158s (_NH); 1743vs (_C═O, COOMe); 1701vs (_C═O, CONH); 1438vs (∂CH, CH₃O); 1223vs (_C—O, COOMe); 864 m (∂ CH, Br═C—H). FAB-MS m/z(assignment): 265/263 (M+H).

EXAMPLE 66 Synthesis of (5-bromouracil)acetic acid

Water (30 mL) was added to the oil of the crude product from Example 65and the mixture was dissolved by adding sodium hydroxide (2 M, 60 mL).After stirring at 0° C. for 10 minutes, hydrochloric acid (4 M, 45 mL)was added to adjust the pH of the solution to 2, and the title compoundprecipitated. After 50 minutes, the solid residue was isolated byfiltration, washed once with cold water, and dried in vacuo oversicapent. Yield: 2.46 g (38%). Mp, 250°-251° C. Anal. for C₆H₅BrN₂O₄.Found (calc.): C, 28.78 (28.94); H, 2.00 (2.02); Br, 32.18 (32.09); N,11.29 (11.25). ¹H-NMR (DMSO-d₆, 250 MHz) δ: 12.55 (1H s, COOH); 11.97(1H, s, NH); 8.30 (1H, s, C═C—H); 4.49 (2H, s, NCH ₂COOH). ¹³C-NMR(DMSO-d₆, 250 MHz) ppm: 169.4 (COOH); 159.8 (NHCOCBr═CH); 150.04 (NCON);145.8 (COCBr═CHN); 94.6 (COCBr═CHN); 48.8 (NCH₂COOH). UV (Methanol;nm_(max)); 226; 278. IR (KBr; cm⁻¹); 3187s (_NH); 1708vs (_C═O,COOH);1687vs; 1654VS (_C═O, CONH); 1192s (_C—O, COOH); 842 m (∂ CH, Br—C═C—H).FAB-MS m/z (assignment, relative intensity); 251/249 (M+H,5).

EXAMPLE 67 Synthesis ofN-(BOC-aminoethyl)-N-(5-bromouracil)methylene-carbonoylglycine ethylester

BOC-aminoethylglycine ethyl ester (1.8 g, 7.30 mmol) was dissolved inDMF (10 mL). Dhbt-OH (1.31 g, 8.03 mmol) was added, whereby aprecipitate was formed. DMF (2×10 mL) was added until the precipitatewas dissolved. The product of Example 66 (2 g, 8.03 mmol) was addedslowly to avoid precipitation. Methylene chloride (30 mL) was added, andthe mixture was cooled to 0° C. and then filtered. The precipitate (DCU)was washed twice with methylene chloride. To the combined filtrate wasadded methylene chloride (100 mL). The mixture was washed with 3×100 mLof half-saturated NaHCO₃-solution (H₂O:saturated NaHCO₃ solution, 1:1,v/v), then with 2×100 mL of dilute KHSQ solution (H₂O:saturated KHSO₄solution, 4:1, v/v), and finally with saturated NaCl solution (1×100mL). The organic phase was dried over magnesium sulphate, filtered, andevaporated to dryness in vacuo (about 15 mm Hg and then about 1 mm Hg).The residue was suspended in methylene chloride (35 mL), stirred for 45minutes at room temperature, and the DCU filtered. Petroleum ether (2volumes) was added dropwise to the filtrate at 0° C., whereby an oilprecipitated. The liquor was decanted and the remaining oil dissolved inmethylene chloride (20-50 mL). Precipitated was effected by the additionof petroleum ether (2 volumes). This procedure was repeated 5 timesuntil an impurity was removed. The impurity can be seen observed by tlcwith 10% MeOH/CH₂Cl₂ as the developing solvent. The resulting oil wasdissolved in methylene chloride (25 mL) and evaporated to dryness invacuo, which caused solidification of the title compound. Yield: 2.03 g((58%). Mp. 87°-90° C. Anal. for C₁₇H₂₅BrN₄O₇. Found (calc.): C, 42.33(42.78); H, 5.15 (5.28); Br, 17.20 (16.74); N, 1.69 (11.74). ¹H-NMR(DMSO-d₆, 250 MHz, J in Hz) δ: 1.93 & 11.92 (1H, s, C═ONHC═O); 8.09 &8.07 (1H, s, C═C—H); 7.00 & 6.80 (1H, t, BOC—NH); 4.80 & 4.62 (2H, s,NCH ₂CON); 4.35 & 4.24 (2H, s, NCH ₂COOEt); 4.27-4.15 (2H, m, COOCH₂CH₃O); 3.47-3.43 (2H, m, BOC—NHCH₂CH ₂N); 3.28-3.25 & 3.12-3.09 (2H, m,BOC—NHCH ₂CH—₂N): 1.46 & 1.45 (9H, s, t-Bu); 1.26 & 1.32 (3H, t, J=7.1;COOCH₂CH ₃). ¹³C-NMR (DMSO-d₆, 250 MHz) ppm: 169.3 & 169.0 (t-BuOC═O);167.4 & 167.1 (COOEt); 159.8 (C═C—CON); 155.9 (NCH₂ CON); 150.4 (NCON);145.9 (COCBr—CHN), 94.5 (COCBr═CHN); 78.2 (Me₃ C); 61.3 & 60.7(COCH₂CH₃); 49.1 & 48.0 (N₂ CH COOH); 48.0 & 47.0 (₂NCH CON); 38.6(BocNHCH₂ CH₂N); 38.2 (BocNHCH₂CH₂N); 26.3 (C(C ₃H₃)); 14.1 (CO₂CH₂ CH).UV (Methanol; _(max) NM): 226; 280. IR (KBr, CM⁻¹): 3200 ms, broad(_NH); 168vs, vbroad (_C═O, COOH, CONH); 1250s (_C—O, COOEt); 1170s(_C—O, COOt-Bu); 859m (∂ CH, Br—C═C—H). FAB-MS m/z (assignment, relativeintensity): 479/477 (M+H, 5); 423/421 (M+2H−t-Bu, 8); 379/377 (M+2H−Boc,100); 233/231 (M-backbone, 20).

EXAMPLE 68 Synthesis ofN-(BOC-aminoethyl)-N-(5-bromouracyl-N¹-methylene-carbonyl)glycine

The product of Example 67 (1.96 g, 4.11 mmol) was dissolved in methanol(30 mL) by heating, and then cooled to 0° C. Sodium hydroxide (2 M, 30mL) was added, and the mixture stirred for 30 minutes. HCl (1 M, 70 mL)was added pH 2). The water phase was extacted with ethyl acetate (3×65mL+7×40 mL). The combined ethyl acetate extracts were washed withsaturated NaCl solution (500 mL). The organic phase was dried overmagnesium sulphate, filtered and evaporated to dryness in vacuo. Yield:1.77 g (96%). Mp. 92°-97° C. Anal. for C₁₅H₂₁BrN₄O₇. Found (calc.): C,40.79 (40.10); H, 5.15 (4.71); Br, 14.64 (17.70); N, 11.35 (12.47).¹H-NMR (DMSO-d₆, 250 MHz, J in Hz) δ: 12.83 (1H, s, COOH); 11.93 & 11.91(1H s, C═ONHC═O); 8.10 & 8.07 (1H, s, C═C—H); 7.00 & 6.81 (1H, t,BOC—NH); 4.79 & 4.61 (2H, s, NCH ₂CON); 4.37 & 4.25 (2H, s, NCH ₂COOH);3.46-3.39 (2H, m, BOC—NHCH₂CH ₂N); 3.26-3.23 & 3.12-3.09 (2H, m,BOC—NHCH ₂CH₂N); 1.46 (9H, s, t-Bu). ^(—C-NMR) 9DMSO-d₆, 250 MHz) ppm:170.4 (t-BuOC═O); 166.9(COOH); 159.7 (C═C—CON); 155.8 (NCH₂ CON); 150.4(NCON); 145.9 (COCBr═CHN); 94.4 (COCBr═CHN); 78.1 (Me₃ C); 49.1 & 48.0(NCH₂COOH); 47.7 & 47.8 (NCH₂CON); 38.6 (BOC—NHC₂ CH₂N); 38.1(BOC—NHCH₂CH₂N); 28.2 (C(CH₃)₃). UV (Methanol; nm_(max)); 226; 278. IR(KBr,cm⁻¹): 3194 ms, broad (_NH); 1686vs, vbroad (_C═O COOH, CONH);1250s (_C—O,COOH); 1170s (_C—O,COOt-Bu); 863m (∂ CH, Br—C═C—H). FAB-MSm/z (assignment, relative intensity): 449/451 (M+H, 70); 349/351(M+2H−BOC, 100); 231/233 (M-backbone, 20).

EXAMPLE 69 Synthesis of uracil-N¹-methyl acetate

Uracil (10 g, 89.2 mmol) and potassium carbonate (24.7 g, 178 mmol) weresuspended in DMF (250 mL). Methyl bromoacetate (8.45 mL, 89.2 mmol) wasadded over a period of 5 minutes. The suspension was stirred overnightunder nitrogen at room temperature, and then filtered. Thin-layerchromatography (10% methanol in ethylene chloride) indicated incompleteconversion of uracil. The solid residue was washed twice with DMF, andthe combined filtrates were evaporated to dryness in vacuo. Theprecipitate was suspended in water (60 mL) and HCl (2.5 mL, 4 M) wasadded (pH 2). The suspension was stirred for 30 minutes at 0° C., andthen filtered. The precipitated title compound was washed with water anddried, in vacuo, over sicapent. Yield: 9.91 g (60%). Mp. 182-183° C.Anal. for C₆H₈N₂O₄. Found (calc.): C, 45.38 (45.66); H, 4.29 (4.38); N,15.00 (15.21). ¹H-NMR (DMSO-d-₆, 250 MHz, J in Hz) δ: 1.47 (1H, s, NH);7.68 (1H, d, I_(H—C═C—H)=7.9), CH═CHN); 5.69 (1H, d, J_(H—C═C—H)=7.9,CH═CHN); 4.59 (2H, s, NCH ₂COOMe); 3.76 (3H, s, COOCH ₃). ¹³C-NMR(DMSO-d₆, 250 MHz) ppm: 168.8 (COOMe); 164.0 (C═C—CON); 151.1 (NCON);146.1 (COCH═CHN); 101.3 (COCH═CHN); 52.5 (COOCH₃); 48.7 (NCH₂COOMe). UV(Methanol; nm_(max)): 226; 261. IR (KBr, cm⁻¹); 3164s (_NH); 1748vs(_C═O, COOMe); 1733vs (_C═O, CONH); 1450vs (∂ CH, CH₃O); 1243VS(_C—O,COOMe); 701m (∂CH, H—C═C—H). FAB-MS m/z (assignment), 185 (M+H).

EXAMPLE 70 Synthesis of Uracilacetic Acid

Water (90 mL) was added to the product of Example 69 (8.76 g, 47.5mmol), followed by sodium hydroxide (2 M, 40 mL). The mixture was heatedfor 40 minutes, until all the methyl ester has reacted. After stirringat 0° C. for 15 minutes, hydrochloric acid (4 M, 25 mL) was added (pH2). The title compound precipitated and the mixture was filtered after2-3 h. The precipitate was washed once with the mother liquor and twicewith cold water and dried in vacuo over sicapent. Yield: 6.66 g (82%).Mp. 288°-289° C. Anal. for C₆H₆N₂O₄. Found (calc.): C, 42.10 (42.36); H,3.43 (3.55); N, 16.25 (16.47). ¹H-NMR (DMSO-d₆), 250 MHz, J in Hz) δ:13.19 (1H, s, COOH); 11.41 (1H, s, NH); 7.69 (1H, d, J_(H—C═C—H)=7.8,J_(H—C—C—N—H)=2.0, COCH═CHN); 4.49 (2H, s, NCH ₂COOH). ¹³C-NMR(DMSO-d-₆, 2509 MHz) ppm: 169.9 (COOH); 163.9 (CH═CHCON); 151.1 (NCON);146.1 (COCH═CHN); 100.9 (COCH═CHN); 48.7 NCH ₂COOH. UV (Methanol;nm_(max)): 246; 263. IR (KBr; cm⁻¹): 3122s (_NH); 1703vs (_C═O, COOH);1698vs, 1692vs (_C═O, CONH); 1205s (_C—O,COOH); 676 (∂ CH, H—C═C—H).FAB-MS m/z (assignment): 171 (M+H).

EXAMPLE 71 Synthesis ofN-(BOC-aminoethyl)-N-(uracil-N¹-methylene-carbonyl)glycine ethyl ester

(BOC-aminoethyl)glycine ethyl ester (2 g, 8.12 mmol) was dissolved inDMF (10 mL). Dhbt-OH (1.46 g, 8.93 mmol) was added and a precipitate wasformed. DMF (2×10 mL) was added until all was dissolved. The product ofExample 70 (1.52 g, 8.93 mmol) was added slowly to avoid precipitation.Methylene chloride (30 mL) was added and the mixture was cooled to 0°C., after which DDC (2.01 g, 9.74 mmol) was added. The mixture wasstirred for 1 h at 0° C., at 2 h at room temperature, and then filtered.The precipitated DCU was washed twice with methylene chloride. Tocombined filtrates was added methylene chloride (100 mL), and thesolution washed with 3×100 mL of half-saturated NaHCO3 solution(H₂O:saturated NaHCO₃solution, 1:1, v/v), then with 2×100 mL of diluteKHSQ solution (H₂O:saturated KHSO₄ solution, 4:1, v/v) and finally withsaturated NaCl solution (1×100 mL). The organic phase was dried overmagnesium sulphate, filtered and evaporated to dryness in vacuo (about15 mm Hg and then about 1 mm Hg). The residue was suspended in methylenechloride (32 mL), and stirred for 35 minutes at room temperature, and 30minutes at 0° C., and then filtered. The precipitate (DCU) was washedwith methylene chloride. Petroleum ether (2 volumes) was added dropwiseto the combined filtrate at 0° C., which caused separation of an oil.The mixture was decanted, the remaining oil was then dissolved inmethylene chloride (20 mL), and then again precipitated by addition ofpetroleum ether (2 volumes). This procedure was repeated 5 times untilan impurity was removed. The impurity can be seen by tlc with 10%MeOH/CH₂Cl₂ as the developing solvent. The resulting oil was dissolvedin methylene chloride (20 mL) and evaporated to dryness in vacuo, whichcaused solidification of the title compound. Yield: 1.71 g (53%). Mp.68.5°-75.7° C. Anal for C₁₇H₂₆N₄O₇. Found (calc.): C, 50.61 (51.25); H,6.48 (6.58); N, 13.33 (14.06). ¹H-NMR (DMSO-d₆, 250 MHz, J in Hz) δ:11.36 (1H, s, C═ONHC═O); 7.51 & 7.47 (1H, d, J_(H—C═C—H)+6.1; COCH═X—H);7.00 & 6.80 (1H, t, BOC—NH); 5.83 & 5.66 (1H, d, J_(H—C═C—H)=5.7,COCH═CH); 4.78 & 4.60 (2H, s, NCH ₂CON); 4.37 & 4.12 (2H, s, NCH₂COOEt); 4.30-4.15 (2H, m, COOCH ₂CH₃); 3.49-3.46 (2H, m, BOC—NHCH₂CH₂n); 3.27 3.23 & 3.11-3.09 (2H, m, BOC—NHCH ₂CH₂N; 1.46 (9H, s, t-Bu);1.39-1.23 (3H, m, COOCH₂CH ₃). ¹³C-NMR (DMSO-d-₆, 250 MHz) ppm: 169.4 &169.0 (t-BuOC═O); 167.6 & 167.3 (COOEt); 163.8 (CH═CHCON); 155.8 (NCH₂CON); 151.0 (NCON); 146.3 (COCH═CHN); 100.8 (COCH═CHN); 78.1 (Me₃ C);61.2 & 60.6 (COOCH₂CH₃); 49.1 (NCH₂COOEt); 47.8 & 47.0 (NCH₂CON); 38.6(BOC—NHCH₂ CH₂N); 38.1 & 37.7 (BOC—NHCH₂N); 28.2 (C(CH₃)₃); 14.1(CO—OCH₂ CH₃. UV (Methanol; _(max) nm); 226; 264. IR (KBr; cm⁻¹): 3053m(_NH); 1685vs, vbroad (_C═O, COOH, CONH); 1253s (_C—O, COOEt); 1172s(_C—O, COOt-Bu); 718w (∂ CH C—C—C—H), FAB-MS m/z (assignment, relativeintensity); 399 (M+H, 35); 343 (M+2H−t-Bu, 100); 299 (M+2H−BOC, 100);153 (M-backbone, 30).

EXAMPLE 72 Synthesis ofN-(BOC-aminoethyl)-N-(uracilmethylene-carbonyl)glycine

The product of Example 71 (1.56 g, 3.91 mmol) was dissolved in methanol(20 mL) and then cooled to 0° C. Sodium hydroxide (2 M, 20 mL) wasadded, and the mixture was stirred for 75 minutes at 0° C. Hydrochloricacid (1 M, 46 mL) was added (pH 2). The water phase was extracted wasethyl acetate (3×50 mL+7×30 mL). The combined ethyl acetate extractswere washed with saturated NaCl solution (360 mL). The organic phase wasdried over magnesium sulphate, filtered, and evaporated to dryness, invacuo. The residue was dissolved in methanol and evaporated to dryness,in vacuo. Yield: 0.55 g (38%). Mp 164°-170° C. Anal. for C₁₅H₂₂N₄O₇.Found (calc.): C, 46.68 (48.65); H, 6.03 (5.99); N, 1461 (15.13). ¹H-NMR(DMSO-d₆, 250 MHz, J in Hz) δ: 12.83 (1H, s, COOH); 11.36 (1H, s,C═ONHC═O); 7.52-7.45 (1H, m, COCH═CHN); 7.00 & 6.82 (1H, t, BOC—NH);5.67-5.62 (1H, m, COCH═CHN); 4.76 & 4.58 (2H, s, NCH₂CON); 4.26 & 4.05(2H, s, NCH ₂COOH); 3.46-3.39 (2H, m, BOCNHCH₂CH ₂N); 3.25-3.23 &3.15-3.09 (2H, m, BOCNHCH ₂CH₂N); 1.46 (9H, s, t-Bu). ¹³C-NMR (DMSO-d₆,250 MHz) ppm: 170.5 (t-BuOC═O); 167.2 (COOH); 163.9 (C═C—CON); 155.8(NCH₂ CON); 151.1 (NCON); 146.4 (COCH═CHN); 100.8 (COCH═CHN); 78.1 (Me₃C); 49.1 & 47.8 (NCH₂ COOH); 47.6 & 46.9 (NCH₂CON); 38.6 (BOC—NHCH₂CH₂N); 38.1 & 37.6 (BOC—NHCH₂CH₂N); 28.2 (C(CH₃)₃). UV (Methanol; _(max)nm); 226; 264. IR (KBr; cm⁻¹); 3190 (_NH); 1685vs, vbroad (_C═O, COOH,CONH); 1253s (_C—O, COOH); 1171s (_C—O, COOt-Bu); 682w (∂ CH, H—C═C—H).FAB-MS m/z (assignment, relative intensity): 371 (M+H, 25); 271(M+H−Boc, 100).

EXAMPLE 73 Synthesis of H-U10-LysNH₂

Synthesis of the title compound was accomplished by using the followingprotocol: (1) BOC-deprotection with TFA/CH₂Cl₂ (1:1, v/v), 3×1 minuteand 1×30 minutes; (2) washing with CH₂Cl₂, 6×1 minute; (3)neutralization with DIEA/CH₂Cl₂ (1:19, v/v), 3×2 minutes; (4) washingwith CH₂Cl₂, 6×1 minute, and drain for 1 minute; (5) at some stages ofthe synthesis, 2-5 mg sample of PNA-resin was removed and driedthoroughly for a ninhydrin analysis to determine the substitution; (6)addition of BOC-protected PNA monomer (free acid) in DMF followed byaddition of DCC in CH₂Cl₂; the coupling reaction was allowed to proceedfor a total of 24 h with shaking; (7) washing with DMF, 1×2 minutes; (8)washing with CH₂Cl₂, 4×1 minute; (9) neutralization with DIEA/CH₂Cl₂(1:19, v/v), 2×2 minutes; (10) washing with CH₂Cl₂, 6×1 minute; (11)occasionally, 2-5 mg sample of protected PNA-resin was removed and driedthoroughly for a ninhydrin analysis to determine the extent of coupling;(12) at some stages of the synthesis, unreacted amino groups wereblocked by acetylation with a mixture of aceticanhydride/pyridine/CH₂Cl₂ (1:1:2, v/v/v) for 2 h followed by washingwith CH₂Cl₂, 6×1 minute, and, occasionally, ninhydrin analysis.

The synthesis was initiated on approximately 100 mg ofLys(ClZ)-MHBA-resin. The crude product (12 mg) was pure enough forhybridization studies. The hybrid between 5′-(dA)10 and H-U10 had T_(m)of 67.5° C.

EXAMPLE 74

Enhanced Stability of Duplex Between PNA Containing 2,6-Diaminopurineand DNA

Four PNA decamers (H-GTAGATCACT-LysNH₂, H-GTAGDTCACT-LysNH₂,H-GTDGDTCDCT-LysNH₂ and H-AGTGATCTAC-LysNH₂; D is 2,6-diaminopurine)were synthesized according to procedures described above. The PNAdecamers were allowed to hybridize with complementary DNA and thethermal melting profiles of the PNA:DNA duplexes were measured andcompared to control DNA:DNA duplexes and with PNA:DNA duplexes whereinthe DNA contains a single TC mismatch opposite a D residue in the PNA.The results are shown in the table below.

*DNA-1 *DNA-2 *PNA-1 (T_(m) in ° C.) (T_(m) in ° C.) (T_(m) in ° C.)PNA-2 51 ˜33 68 H-GTAGATCACT-LysNH₂ (SEQ ID NO:1) PNA-3 56 32.5 71.5H-GTAGDTCACT-LysNH₂ (SEQ ID NO:2) PNA-4 67 55.5 81 H-GTDGDTCDCT-LysNH₂(SEQ ID NO:3) DNA-3 33.5 **ND 49 5′-dGTAGATCACT-3′ (SEQ ID NO:4) DNA-436 28 57 5′-dGTAGDTCACT-3′ (SEQ ID NO:5) DNA-5 44 34.5 615′-dGTDGDTCDCT-3′ (SEQ ID NO:6) *DNA-1 is 5′-dAGTGATCTAC-3′ (SEQ IDNO:7) DNA-2 is 5′-dAGTGACCTAC-3′ (SEQ ID NO:8) PNA-1 isH-AGTGATCTAC-LysNH₂ (SEQ ID NO:9) **ND = Not Determined

The presence of 2,6-diaminopurine increased the Tm by 4-5° C. in PNA:DNAduplexes whereas the increase in DNA:DNA duplexes was only 3-4° C. Thisdifference may reflect increased stacking in the former duplex. Thistendency is also observed when unmodified homoduplexes of PNA:PNA andDNA:DNA are compared to duplexes having three D:T base pairs, resultingin T_(m) increases of 12° C. and 10.50° C., respectively. Uponcomparison of the binding of PNA-3 to DNA-1 and DNA-2, a decrease inT_(m) of 23.5° C. is observed when a D:C mismatch is introduced into aPNA:DNA duplex containing only one 2,6-diaminopurine nucleobase, whereasa decrease in T_(m) of only 18° C. is observed in case of PNA-2 whichdoes not contain any 2,6-diaminopurine nucleobases. The increasedspecificity in terms of a decrease in the melting temperature (T_(m)) ofthe duplex was not observed when additional D:T basepairs wereintroduced. This finding reflects the increased stability of the duplexcontaining more than a single 2,6-diaminopurine nucleobase indicatingthat the more stable duplex is better able to accomodate themismatch-induced structural change in the duplex.

EXAMPLE 75

Binding of PNA Containing 2,6-Diaminopurine to DNA

A PNA decamer containing six 2,6-diaminopurine nucleobases was allowedto hybridize to complementary DNA The thermal stability of this duplexwas determined and compared to thermal stability of a duplex formedbetween DNA and a PNA decamer devoid of 2,6-diaminopurine nucleobases.The T_(m) results are shown in the table below.

*DNA-6 *DNA-7 (T_(m) in ° C.) (T_(m) in ° C.) PNA-5  71 50H-AAAAGGAGAG-LysNH₂ (SEQ ID NO:10) PNA-6 ≧85 71 H-DDDDGGDGDG-LysNH₂ (SEQID NO:11) *DNA-6 is 5′-CTCTCCTTTT-3′ (SEQ ID NO:12) DNA-7 is5′-TTTTCCTCTC-3′ (SEQ ID NO:13)

Incorporation of 2,6-diaminopurine nucleobases into PNA increasesthermal stability (represented by the T_(m)) of the duplex between DNAand the PNA containing a 2,6-diaminopurine nucleobase to 85° C. whencompared with the thermal stability (T_(m)=71° C.) of the duplex betweenDNA and a PNA devoid of 2,6-diaminopurine nucleobases.

EXAMPLE 76 Synthesis ofethyl-N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetate (14, FIG. 7a)

To a suspension of 2,6-diaminopurine (3 g, 19.46 mmol) in dry DMF (90mL) was added NaH (60% in oil 0.87 g, 21.75 mmol). After 1 hour ethylbromoacetate (4.23 g, 25.34 mmol) was added. The reaction mixture becamehomogenous in 30 minutes and was allowed to stir for an additional 90minutes. The DMF was removed in vacuo resulting in a tan powder. The tanpowder was then refluxed with 1,4-dioxane (200 mL) for 10 minutes andfiltered through celite. The solution was concentrated to give a lightyellow powder. To the light yellow powder (5.52 g) in 1,4-dioxane (150mL) was added freshly prepared N-benzyloxycarbonyl-N′-methylimidazoliumtriflate (10.7 g, 29.2 mmol). The reaction mixture was stirred at roomtemperature for 16 h resulting in a reddish solution. The dioxane wasremoved in vacuo and the crude material was recrystallized fromMeOH:diethyl ether to give 4.56 g (63%) of the title compound as acream-colored solid.

¹H NMR (DMSO-d₆) δ: 10.12 (bs, 1H), 7.43 (m, 5H), 6.40 (bs, 2H), 5.17(s, 2H), 4.94 (s, 2H), 4.18 (q, J=7.2, 3H), 1.21 (t, J=7.2, 3H). ¹³C NMR(DMSO-d₆) ppm: 167.81, 159.85, 154.09, 152.07, 149.77, 140.62, 136.42,128.22, 127.74, 127.61, 166.71, 65.87, 61.21, 43.51, 13.91.

EXAMPLE 77 Synthesis ofN6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetic acid (15, FIG. 7 a)

Ethyl-N6-(benzyloxycarbonyl)-2,6diaminopurin-9-yl-acetate (14, 3 g, 8.1mmol) was dissolved in NaOH (2 N, 30 mL). After 1 h the solution wasacidified to pH 2.5 with 2 M HCl. The precipitate was filtered, washedwith water, and dried to give 2.82 g (98%) of the title compound as awhite solid.

IR(KBr): 3300, 3095, 1750, 1630, 1590, 1410. ¹H NMR(DMSO-d₆) δ: 10.11(s, 1H), 7.91 (s, 1H), 7.45-7.33 (m, 5H), 6.40 (s, 2H), 5.17 (s, 2H),4.83 (s, 2H).

EXAMPLE 78 Synthesis of BOC-aminoacetaldehyde (16, FIG. 7 b)

The title compound was prepared according to a published literatureprocedure (Dueholm et al., Organic Preparations and Procedures Intl.,1993, 25, 457).

EXAMPLE 79 Synthesis of lysine(2-chlorobenzyloxy) allyl ester (17, FIG.7 b)

The title compound was prepared according to a published literatureprocedure (Waldmann and Horst, Liebigs Ann. Chem, 1983, 1712).

EXAMPLE 80 Synthesis of N-(BOC-aminoethyl)-Lysine-(2-chlorobenzyloxy)allyl ester (18, FIG. 7 b)

p-Toluenesulphonic acid-protected lysine (11 mmol) was dissolved inCH₂Cl₂ (100 mL) and washed with saturated aqueous NaHCO₃ (100 mL). Theaqueous layer was back-extracted with CH₂Cl₂ and the CH₂Cl₂ layers werecombined, dried over Na₂SO₄, and concentrated to give the free lysine asan oil. The resulting oil was taken up in methanol (50 mL) and cooled to0° C. To the resulting solution was added sodium cyanoborohydride (5.9mmol) followed by acetic acid (0.75 mL). After 5 minutesBOC-aminoacetaldehyde (13.3 mmol) was added and the reaction mixture wasstirred for an additional 1 h. The methanol was removed in vacuo and theoil was dissolved in ethyl acetate (40 mL), washed with saturatedaqueous NaHCO₃, brine, dried over Na₂SO₄ and concentrated to give aclear colorless oil. This oil was dissolved in dry ether (80 mL), cooledto −20° C., and a molar equivalent of HCl in ether was added slowly. Theresulting white solid was collected by filtration and air dried.Precipitation of the air-dried white solid from dry ether gaveanalytically pure title compound.

EXAMPLE 81 Synthesis ofN-(BOC-aminoethyl)-N-[N⁶-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetyl]-Lysine-(2-chlorobenzyloxy)allyl ester (19, FIG. 7 b)

To N⁶-(benzyloxycarbonyl)2,6-diaminopurin-9-yl-acetic acid (15, 3.6 g,10.5 mmol) in DMF (150 mL) was added N,N-diisopropylethylamine (2.75 mL,21 mmole), and N-(BOC-aminoethyl)-lysine-(2-chlorobenzyloxy) allyl esterhydrochloride (7.31 gm, 15.8 mmol). The reaction mixture was stirredunder nitrogen for 20 minutes and bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBrop, 5.4 gm, 11.6 mmol) was added. The reactionmixture was stirred overnight at room temperature under an atmosphere ofnitrogen gas. The resulting mixture was concentrated and dissolved inethyl acetate. The ethyl acetate solution was washed with aqueoussaturated sodium bicarbonate, separated and concentrated. The crudematerial was purified by silica gel flash column chromatography usingethyl acetate:hexane: methanol (6:3:1, v/v/v), as the eluent.Concentration and drying of the appropriate fractions gave 3.1 g (37%)of the title compound.

EXAMPLE 82 Synthesis ofN-(BOC-aminoethyl)-N-[N⁶-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetyl]-Lysine-(2-chlorobenzyloxy)(20, FIG. 7 c)

ToN-(BOC-aminoethyl)-N-[N⁶-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetyl]-lysine-(2-chlorobenzyloxy)allyl ester hydrochloride (19, 3.1 gm, 3.93 mmol) was added THF (100 mL)morpholine (3.5 mL, 39.3 mmol), andtetrakis(triphenylphosphine)-palladium(0) (0.45 gm, 0.393 mmol). Thereaction mixture was stirred under an atmosphere of nitrogen for 2.5 hat room temperature. The resulting mixture was concentrated anddissolved in ethyl acetate. The ethyl acetate solution was washed withaqueous saturated potassium hydrogen sulfate (that was half-diluted withwater), separated and concentrated. The crude material was purified bysilica gel flash column chromatography using chloroform:methanol (9:1,v/v), as the eluent. Concentration and drying of the appropriatefractions gave 1.25 g (42%) of the title compound.

EXAMPLE 83

Standard Protocol For PNA Synthesis and Characterization

Instrument: PerSeptive Biosystems 8909 Expedite.

Synthesis Scale: 2 μmole.

Reagents:

Wash A: 20% DMSO in NMP

Wash B: 2 M Collidine in 20% DMSO in NMP

Deblock: 5% m-Cresol, 95% TFA

Neutralizer: 1 M DIEA in 20% DMSO in NMP

Cap: 0.5 M Acetic Anhydride, 1.5 M Collidine in 20% DMSO in NMP

Activator: 0.2 M HATU in DMF

Monomers: 0.22 M in 2 M Collidine (50% Pyridine in DMF)

Synthesis: The solid support (BOC-BHA-PEG-resin) is washed with 708 μLof Wash A. Deblock (177 μL) is passed through the column 3 times over6.3 minutes. The resin is then washed with 1416 μL of Wash A. The freeamine is neutralized with 1063 μL of Neutralizer. The resin is washedwith 1062 μL of Wash B. Monomer and Activator (141 μL each) are slowlyadded to the column over 14 minutes. The resin is washed with 708 μL ofWash B and 708 μL of Wash A. Unreacted amine is capped with slowaddition of 708 μL of Cap solution over 5 minutes. The resin is thenwashed 2124 μL of Wash A. The cycle is repeated until synthesis of thedesired PNA sequence is completed.

Cleavage: The PNA-resin is washed with 5 mL of MeOH and dried undervacuum. The dried resin is emptied into a 1.5 mL Durapore ultrafreefilter unit. Thioanisole (25 μL), 25 μl of m-Cresol, 100 μL of TFA and100 μL of TFMSA is added to the resin, vortexed for about 30 seconds andallowed to stand for 2 h. The reaction mixture is then centrifuged for 5minutes at 10 K and the inner tube with resin is removed. Approximately1.5 mL of ether is added to the TFA solution to precipitate the product.The TFA solution is vortexed, followed by centrifugation at 10 K for 2minutes. The ether is removed in vacuo. Ether precipitation andcentrifugation are repeated an additional 2 times. The dry pellet isheated in a heat block (55° C.) for 15 to 30 minutes to remove excessether and redissolved in 200 μL of H₂O. Solvent is added to 100 mg ofDowex Acetate Resin in a 1.5 mL Durapore ultrafree filter unit,vortexed, allowed to stand for 30 minutes and centrifuged at 10 K for 2minutes.

Characterization: The absorbance of a 1 μL sample in 1 mL of H₂O ismeasured at 260 nm. Isopropanol (50%) in H₂O with 1% Acetic acid (100μL) is added to 4 μL of the sample. This sample is characterized byelectrospray mass spectrometry.

Common Abreviations

NMP: N-methyl pyrrolidinone

TFA: Trifluoroacetic acid

DIEA: N,N-Diisopropylethylamine

HATU: O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate

TFMSA: Trifluormethanesulfonic Acid

EXAMPLE 84

PNA Oligomers Containing 2,6-diaminopurine Attached to anAminoethyl-Lysine Backbone

Using the title compound of Example 82, the aminoethylglycine PNAmonomers of examples 24 through 34, and the standard protocol for PNAsynthesis illustrated in Example 83, the following PNA oligomers wereprepared:

SEQ ID NO:14 TTT-CGC-GDkC-CCDk SEQ ID NO:15 GCDk-DkDkC-GC

C, G, and T are nucleobases cytosine, guanine, and thymine respectively,attached to an aminoethylglycine PNA backbone. Dk is 2,6-diaminopurineattached to an aminoethyl-lysine backbone as illustrated in the previousexamples. Aminoethyl-lysine backbone is an aminoethyl-glycine backbonewith butylamine substituent at a-position, i.e., lysine side-chain.

EXAMPLE 85

Synthesis of PNA Oligomers Having at Least One A, G, C, or T Attached toa Lysine-containing Backbone

Using the procedures of Example 83, the aminoethylglycine PNA monomersof examples 24 through 34, and monomers of Examples 76-82, the followingPNA oligomers were synthesized (Tk is thymine attached to anaminoethyl-lysine backbone; Gk is guanine attached to anaminoethyl-lysine backbone; Ck is cytosine attached to anaminoethyl-lysine backbone):

SEQ ID NO:16 CGC-TkTkG-GCA-GTkC-TkC SEQ ID NO:17CGkC-TkTkGk-GkCA-GkTkC-TkC SEQ ID NO:18 CkGkCk-TkTkG-GkCkA-GkTkCk-TkCkSEQ ID NO:19 TkTkTk-AGG-ATkTk-CGTk-GCTk-C SEQ ID NO:20TkCG-TkGC-TkCA-TkGG SEQ ID NO:21 GCG-TkTkTk-GC SEQ ID NO:22CGC-TkGC-AGA-TkGC-GGTk-Tk SEQ ID NO:23 CCG-CCG-GCTk-CAG-TkCTk-Tk SEQ IDNO:24 CATk-CGTk-GGC-GGTk-TkAG-G SEQ ID NO:25 TkCG-GGTk-GAG-TkGG-TkAG SEQID NO:26 CAC-TkCA-GTkG-CAA-CTkC-Tk SEQ ID NO:27 CCTk-CCA-CTkC-CCG-CCTk-CSEQ ID NO:28 CkATk-CkGTk-GGCk-GGTk-TkAG-G SEQ ID NO:29CAC-TkCA-GTkG-CAA-CTkC-Tk SEQ ID NO:30 CCTk-CCA-CTkC-CCG-CCTk-C SEQ IDNO:31 CAGk-CCA-TkGG-TTkC-CCC-CkCA-AC SEQ ID NO:32Fla-GTkG-AGG-GTkC-TkCTk-CTC SEQ ID NO:33 Cy5-GTkG-AGG-GTkC-TkCTk-CTC SEQID NO:34 Fla-CAA-ATkG-GTkTk-CTkC-GAA SEQ ID NO:35Cy5-CAA-ATkG-GTkTk-CTkC-GAA SEQ ID NO:36 Fla-ACC-TGkA-GkGGk-AGkC-CAG SEQID NO:37 Cy5-ACC-TGkA-GkGGk-AGkC-CAG SEQ ID NO:38Fla-TkTkG-GCC-ACG-TkCC-TkGA SEQ ID NO:39 Cy5-TkTkG-GCC-ACG-TkCC-TkGA SEQID NO:40 Fla-TGkC-CCG-GkGkA-AAA-CGkT SEQ ID NO:41Cy5-TGkC-CCG-GkGkA-AAA-CGkT SEQ ID NO:42 Fla-CCTk-CGTk-GCA-CGTk-TkCTkSEQ ID NO:43 Cy5-CCTk-CGTk-GCA-CGTk-TkCTk SEQ ID NO:44Fla-TkGG-ATkG-TkCG-ACC-TkCTk

EXAMPLE 86 Synthesis of methyl a-formylsuccinate

This procedure is a modification of published method (Fissekis et al.,Biochemistry, 1970, 9, 3136). Sodium methoxide (40.5 g, 0.75 mol) wassuspended in dry ether (500 mL) and stirred under nitrogen at 0° C. Amixture of dimethylsuccinate (65.4 mL, 0.5 mol) and methylformate (123mL, 2 mol) was added dropwise over 30 minutes. The reaction mixture wasstirred at 0° C. for 2 h and then at room temperature overnight.Subsequently, the reaction mixture was evaporated to a viscous brownresidue which was washed once with petroleum ether and then dissolved in3 M hydrochloric acid (160 mL). This solution was made weakly acidicwith concentrated hydrochloric acid and then extracted withdichloromethane (4×250 mL). The organic phase was dried (MgSO₄),filtered and evaporated under reduced pressure. The resulting residuewas distilled in a kugelrohr apparatus at 60° C. and 0.6 mBar yielding52.3 g of a mixture of the title compound and dimethyl succinate in themolar ratio 80:20 (determined by NMR) as a colorless oil. The product ispurified of the dimethyl succinate by continuous extraction with diethylether. Alternatively the mixture can be used directly for the next step.

¹H NMR (DMSO-d₆, TMS) δ: 3.2 (s, 2H, CH₂), 3.59 (s, 3H, OMe), 3.61 (s,3H, OMe), 7.73 (s, 1H, CHOH), 10.86 (br s, 1H, CHOH). ¹³C NMR (DMSO-d₆,TMS) ppm: 28.9 (CH₂), 51.0 (OMe), 51.6 (OMe), 102.1 (C═CHOH), 156.6(CHOH), 168.3 (COO), 171.7 (COO).

EXAMPLE 87 Synthesis of pseudoisocytosine-5-ylacetic acid

This procedure is a modification of a published method (Beran et al.,Collect. Czech. Chem. Commun., 1983, 48, 292). Sodium methoxide (41.9 g,0.78 mol) was dissolved in dry methanol (200 mL) and guanidinehydrochloride (49.4 g, 0.52 mol) was added. The mixture was stirred for10 minutes under nitrogen at room temperature. A solution of methylα-formylsuccinate (30 g, 0.17 mol) in dry methanol (100 mL) was added tothe mixture. The reaction mixture was refluxed under nitrogen for 3 hand then stirred at room temperature overnight. The reaction mixture wasfiltered, and the filtered residue washed once with methanol. Thecollected filtrate and washing were evaporated under reduced pressure.The resulting residue was dissolved in water (80 mL) and the solutionwas acidified with concentrated hydrochloric acid to pH 4.2. Afterhaving been stirred at 0° C. the mixture was filtered, the precipitatewashed once with water and then freeze-dried leaving 28.29 g (97%) ofthe title compound as a white solid.

Anal. Calcd for C₆H₇N₃O₃ 1/2 H₂O: C, 40.45; H, 4.53; N, 23.59. Found: C,40.13; H, 4.22; N, 23.26. Due to the poor solubility properties of theproduct it was further characterized as its sodium salt. The titlecompound (0.42 g, 2.5 mmol) and sodium bicarbonate were dissolved inboiling water (35 mL). The solution was cooled and evaporated. Theresidue was dissolved in water (6 mL) and ethanol (4 mL) and isopropanol(8 mL) were added. The sodium salt was collected by filtration, washedwith absolute ethanol and petroleum ether and dried to yield 0.31 g ofthe product (65%) as white crystals.

¹H NMR (D₂O, TMS) δ: 3.10 (s, 2H, CH₂COO), 7.40 (s, 1H, H6). ¹³C NMR(DMSO-d₆, TMS) ppm: 34.8 (CHCOO), 112.0 (C-5), 145.6-146.5 (m, C-2),155.1 (C-6), 169.4 (C-4), 179.3 (COOH). MS (FAB) m/z (%): 192 (100,M+H).

EXAMPLE 88 Synthesis of methyl pseudoisocytosin-5-yl acetate

Thionylchloride (3.6 mL, 50 mmol) was added to stirred methanol (210 mL)at −40° C. under nitrogen. Pseudoisocytosin-5-ylacetic acid (7 g, 41mmol) was added and the reaction mixture was stirred at room temperaturefor 1 hour at 60° C., and overnight at room temperature. The reactionmixture was evaporated to dryness and the residue was dissolved insaturated aqueous sodium bicarbonate (80 mL) giving a foamy precipitate.4 M Hydrochloric acid was added (solution pH 6.5) and the suspension wasstirred for 1 h. The precipitate was collected by filtration, washedwith water, recrystallized from water and freeze-dried yielding 4.66 g(62%) of methyl isocytosin-5-ylacetate as white crystals.

¹H NMR (DMSO-d₆, TMS) δ: 3.28 (s, 2H, CH₂COO), 3.64 (s, 3H, COOMe), 6.87(br s, 2H, NH₂), 7.54 (s, 1H, H-6). ¹³C NMR (DMSO-d₆, TMS) ppm: 32.0 (CH₂COO), 51.5 (COOMe), 108.4 (C-5), 153.3 (C-2), 156.4 (C-6), 164.0 (C-4),171.8 (CH₂ COO). MS (FAB+) m/z (%): 184 (100, M+H). Anal. Calcd forC₇H₉N₃O₃ 3/2 H₂O: C, 40.00; H, 5.75; N, 19.99. Found: C, 40.18; H, 5.46;N, 20.30.

EXAMPLE 89 Synthesis of methylN2-(benzyloxycarbonyl)pseudoisocytosin-5-yl acetate

Methyl pseudoisocytosin-5-ylacetate (9.5 g, 52 mmol) was dissolved indry DMF (95 mL) and the solution was stirred at 0° C. under nitrogen.N-benzyloxycarbonyl-N′-methylimidazolium triflate (37.99 g, 104 mmol)was added slowly. The reaction mixture was stirred for 30 minutes at 0°C. and then overnight at room temperature. Dichloromethane (800 mL) wasadded and the resultant mixture was washed with half-saturated aqueoussodium bicarbonate (2×400 mL), half-saturated aqueous potassium hydrogensulfate (2×400 mL) and brine (1×400 mL). The organic phase was dried(MgSO₄), filtered and evaporated under reduced pressure. The residue wasrecrystallized from methanol affording 13.32 g (81%) of the titlecompound as white crystals.

¹H NMR (DMSO-d₆, TMS) δ: 3.43 (s, 2H, CH₂COO), 3.67 (s, 3H, COOMe), 5.30(s, 2H, PhCH ₂), 7.43-7.52 (m, 5H, PhCH₂), 7.77 (s, 1H, H-6). ¹³C NMR(DMSO-d₆, TMS) ppm: 31.9 (CH ₂COO), 51.6 (COOMe), 67.0 (PhCH ₂),128.1-128.5 (m, Ph ₂CH), 135.7 (PhCH₂), 150.7 (Z—CO), 170.8 (COO). MS(FAB+) m/z (%): 318 (3.5, M+H). Anal. Calcd for C₁₅H₁₅N₃O₅: C, 56.78; H,4.76; N, 13.24. Found: C, 56.68; H. 4.79; N, 13.28.

EXAMPLE 90 Synthesis of N2-(benzyloxycarbonyl)pseudoisocytosin-5-ylacetic acid

Methyl N2-(benzyloxycarbonyl)pseudoisocytosin-5-yl acetate (5.2 g, 16mmol) was suspended in THF (52 ml) and cooled to 0° C. 1 M lithiumhydroxide (49 mL, 49 mmol) was added and the reaction mixture wasstirred at 0° C. for 25 minutes. Additional 1 M lithium hydroxide (20mL, 20 mmol) was added and the mixture was stirred at 0° C. for 90minutes. The product was precipitated by acidifying to pH 2 with 1 Mhydrochloric acid, collected by filtration, washed once with water anddried. The yield was 4.12 g (83%) as white crystals.

¹H NMR (DMSO-d₆, TMS) δ: 3.33 (s, 2H, CH₂COO), 5.29 (s, 2H, PhCH₂),7.43-7.52 (m, 5H, PhCH₂), 7.74 (s, 1H, H-6), 11.82 (br s, 3H,exchangeable protons). MS (FAB+) m/z (%): 304 (12, M+H). Anal. calcd.for C₁₄H₁₃N₃O₅: C, 55.45; H, 4.32; N, 13.86. Found: C, 55.55; H, 4.46;N, 13.84.

EXAMPLE 91

Preparation of Pseudoisocytosine Attached to an Aminoethyl LysineBackbone

N2-(benzyloxycarbonyl)pseudoisocytosin-5-ylacetic acid was atttached toN-(BOC-aminoethyl)-lysine-(2-chlorobenzyloxy) allyl ester (18) as perthe procedure of Example 81. The resulting monomeric compound is treatedas per the procedure of Example 82 to give the deprotected compoundready for use in oligomer synthesis.

EXAMPLE 92

Synthesis of PNA Oligomer Having a Pseudoisocytosine Attached to anAminoethyl-lysine Backbone

Aminoethyl-lysine pseudoisocytosine monomer was incorporated into PNAsusing the procedure of Example 83.

EXAMPLE 93

Preparation of PNA Monomers Having Adenine, Guanine, Cytosine, andThymine Attached to an Aminoethyl-lysine Backbone

a) Preparation of the guanine monomer: ToN6-benzyl-9-carboxymethylene-guanine (2.63 g, 8.78 mmol) was added DIEA(2.6 mL, 20 mmol), DMF (30 mL), dichloromethane (70 mL), andN-(BOC-aminoethyl)-lysine-(2-chlorobenzyloxy) allyl ester (18, 3.7 g,8.04 mmol). The reaction mixture was stirred under nitrogen for 20minutes. PyBrop (4 g, 8.58 mmol) was added and the reaction mixturestirred for an additional 16 h. The reaction mixture was concentratedand the residue was purified by silica gel flash column chromatographyusing chloroform/hexanes/methanol (12:7:1, v/v/v) to give 4 g (60%) ofthe title compound as the allyl ester.

To the allyl ester (4 g, 5.37 mmol) was added THF (100 mL), tetrakispalladium(0) (0.18 g, 0.15 mmol), and morpholine (6.1 mL, 70 mmol). Thereaction mixture was stirred under nitrogen for 2.5 h and concentrated.The residue was purified by silica gel flash column chromatography usingchloroform/hexanes/methanol (11:8:1, v/v/v) to give 2.67 g (60%) of thetitle compound.

b) Preparation of the adenine monomer: The procedure used for theguanine monomer in Example 93(a) above was followed for the synthesis ofthe adenine monomer using N6-benzyl-9-carboxymethylene-adenine.

c) Preparation of the cytosine monomer: ToN-(BOC-aminoethyl)-lysine-(2-chlorobenzyloxy) allyl ester (18, 8.21 g,17.7 mmol), added triethylamine (10 mL, 98 mmol) and dichloromethane(200 mL). The solution was cooled to about 0° C. in an ice bath undernitrogen. To the cooled solution was added chloroacetyl chloride (2.2mL, 27.6 mmol) over 10 minutes and the reaction mixture stirred at roomtemperature for 16 h. The reaction mixture was concentrated and theresidue was purified by silica gel flash column chromatography usingethyl acetate/ hexanes (1:1, v/v) to give 6.54 g (68%) of theN-acetylated lysine backbone.

Cytosine is protected at the N4 position by treatment with benzylchloroformate in pyridine at 0° C. to give N4-benzyl-cytosine.

To N⁴-benzyl-cytosine (1.31 g, 5.34 mmol) was added DMF (200 mL), and60% NaH in mineral oil (0.22 g, 5.4 mmol) and the resulting mixture wasstirred under nitrogen for 30 minutes. To the resulting mixture wasadded the N-acetylated lysine backbone (2.9 g, 5.34 mmol) in DMF (25 mL)and the mixture stirred for 16 h. The reaction mixture was concentratedand the residue dissolved in dichloromethane (250 mL). Thedichloromethane phase was washed with water (200 mL) and concentrated.The resulting residue was purified by silica gel flash columnchromatography using dichloromethane:hexanes: methanol (8:2:1) to give2.4 g (85%) of the cytosine attached to the aminoethyl-lysine backboneas the allyl ester.

The allyl ester is converted to the active monomer by deprotection usingpalladium following the procedure used in Example 93(a) above to give1.05 g (46%) of the title compound.

Also see Examples 86-91.

d) Preparation of the thymine monomer: The thymine monomer was preparedfollowing the procedure of Example 93(c) above.

EXAMPLE 94

In vitro Evaluation of PNAs Targeted to HCV

HCV replication in cell culture has not yet been achieved. Consequently,in vitro translation assays are used as standard assays to evaluatecompounds for their anti-HCV activity. One such standard in vitrotranslation assay was used to evaluate PNAs of the present invention fortheir ability to inhibit synthesis of HCV protein in a rabbitreticulocyte assay.

Plasmids containing full-length cDNA sequence for the desired portion ofthe HCV mRNA was prepared. A T7 promoter was introduced into the plasmidimmediately adjacent to the 5′-cap site. A similar strategy was used formaintaining a control in which a cDNA plasmid containing codingsequences for a truncated intercellular adhesion molecule type I wasmodified. As a result of a deletion at base 554 relative to the ICAM-1AUG, a frameshift occurs with a stop codon generated at base 679. Theresulting open reading frame encodes a truncated ICAM-1 polypeptide witha lower molecular weight.

Uncapped transcripts for in vitro translation were prepared by T7transcription of the plasmid using the Megascript transcription kit(Ambion, Inc.) according to the instructions provided by themanufacturer. The plasmid was linearized by restriction endonucleasedigestion at a site in the linker region of the plasmid immediatelydownstream of the 3′-untranslated sequences of the cDNA insert in orderto generate a transcript nearly identical in sequence to authentic mRNA.Following transcription, free nucleotides were removed using G-50Quickspin columns (Boehringer-Mannheim) and the amount of transcriptpresent was quantitated by optical density.

In vitro translation reactions contained 300 ng of the HCV transcript(final concentration of 10 nM), 7 μL of rabbit reticulocyte lysate (RRL,Promega), 8.8 μCi of [³⁵S]-methionine (1175 Ci/mmol, Amersham), 13 μMIVT amino acids mix devoid of methionine (Promega), 8 units of RNasin(Promega) and PNAs in a total volume of 15 μL. A similar controlreaction contained 100 ng (30 nM) of the truncated ICAM-1 transcriptinstead of the HCV transcript. The target and control RNA were heated at65° C. for 5 minutes, incubated at 37° C. for 15 minutes and then mixedwith lysate components. The translation mix was incubated at 37° C. for60 minutes and the reaction was terminated by the addition of 2× Laemmligel loading buffer. After boiling, proteins were fractionated on precast14% acrylamide gels (Novex, San Diego), fixed in 10% propanol, 5% aceticacid, 3% glycerol, dried and analyzed with a PhosphorImager.

PNAs effectively blocked in vitro translation of HCV protein. PNAs thatwere evaluated in an in vitro translation assay are shown in the tablebelow (Dk and Tk are 2,6-diaminopurine and thymine, respectively,attached to an aminoethyl-lysine backbone).

SEQ ID ISIS # NO: SEQUENCE 13642 14 TTT-CGC-CDkC-CCDk 13414 19TkTkTk-AGG-ATkTk-CGTk-GCTk-C 13639 20 TkCG-TkGC-TkCA-TkGG 265-12 45TTT-CGC-GAC-CCA 11908 46 TCG-TGC-TCA-TGG  8215 47Gly-TTT-AGG-ATT-CGT-GCT-CAT-GG-LysCONH₂

Results of the in vitro translation assay are shown in FIG. 8. It isobserved that 13642 (which is a 12-mer PNA containing two2,6-diaminopurine nucleobases bearing lysine side chains) with an EC₅₀of approximately 29 nM is more effective at blocking in vitrotranslation of HCV protein than 265-12 (which is devoid of lysine sidechains) with an EC₅₀ of approximately 57 nM. Further, upon comparing11908 and 13639, at a concentration as low as 30 nM, it is evident thatthe PNA with lysine side chains (i.e. 13639) is more effective atblocking in vitro translation of HCV protein than 11908, which does notcontain any lysine side chains. This clearly indicates that the presenceof a side chain in PNA enhances its ability to block in vitrotranslation of HCV protein.

EXAMPLE 95

Thermal Stability of PNA Duplexes

Duplex-forming PNAs were synthesized as described in Example 85. PNAhaving the sequence H-GTxA-GATx-CAC-Tx-R (SEQ ID NO:1, wherein Txrepresents a thymine monomer bearing an amino acid side chain) wasallowed to hybridize with complementary DNA having the sequence5′-AGT-GAT-CTA-C-3′ (SEQ ID NO:7) and complementary PNA having thesequence H-AGT-GAT-CTA-C-LysNH₂ (SEQ ID NO:9), and the thermalstabilities (T_(m)) of the duplexes were determined in 10 mM phosphate,100 mM NaCl and 1 mM EDTA at a pH of 7. The results are shown in thetable below.

Anti- Anti- parallel Parallel Parallel DNA PNA DNA X* C-Terminal T_(m)(° C.) T_(m) (° C.) T_(m) (° C.) Glycine  a^(#) 52 68 38 Glycine  b^(#)49 67 38 L-Lysine a 52 64 41 D-Lysine a 55 N/D 40 L-Serine a 45 62 37D-Serine a 50 64 38 L-Glutamic b NC N/D NC Acid D-Glutamic b 42 60 −28Acid L-Aspartic b 39 N/D 33 Acid L-Isoleucine a 40 53 NC NC =non-cooperative (no duplex formation occurred) N/D = not determined *Thebackbone at the Tx position bears the indicated amino acid side chain.^(#)In PNA of SEQ ID NO:1, a indicates that R = NH₂; and b indicatesthat R = LysNH₂.

The results show that glycine in the backbone can be replaced by otheramino acids for a moderate loss in hybridization potency. Upon comparingD-lysine versus L-lysine and D-serine versus L-serine, it is evidentthat D-amino acids are better accomodated in the backbone of PNAs.Furthermore, the introduction of a negatively-charged side chain in thePNA backbone (e.g. glutamic acid and aspartic acid) decreaseshybridization potency as indicated by a decreased T_(m), whereas apositively-charged side chain (e.g. lysine) increases the hybridizationpotency as indicated by a higher T_(m). Also, the PNAs bound better toantiparallel DNA than to parallel DNA.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the presentinvention and that such changes and modifications may be made withoutdeparting from the spirit of the invention. It is therefore intendedthat the appended claims cover all such equivalent variations as fallwithin the true spirit and scope of the invention.

60 1 11 DNA Artificial Sequence Synthetic Construct 1 gtagatcact n 11 211 DNA Artificial Sequence Synthetic Construct 2 gtagntcact n 11 3 11DNA Artificial Sequence Synthetic Construct 3 gtngntcnct n 11 4 10 DNAArtificial Sequence Synthetic Construct 4 gtagatcact 10 5 10 DNAArtificial Sequence Synthetic Construct 5 gtagntcact 10 6 10 DNAArtificial Sequence Synthetic Construct 6 gtngntcnct 10 7 10 DNAArtificial Sequence Synthetic Construct 7 agtgatctac 10 8 10 DNAArtificial Sequence Synthetic Construct 8 agtgacctac 10 9 10 DNAArtificial Sequence Synthetic Construct 9 agtgatctac 10 10 11 DNAArtificial Sequence Synthetic Construct 10 aaaaggagag n 11 11 11 DNAArtificial Sequence Synthetic Construct 11 nnnnggngng n 11 12 10 DNAArtificial Sequence Synthetic Construct 12 ctctcctttt 10 13 10 DNAArtificial Sequence Synthetic Construct 13 ttttcctctc 10 14 12 DNAArtificial Sequence Synthetic Construct 14 tttcgcgncc cn 12 15 8 DNAArtificial Sequence Synthetic Construct 15 gcnnncgc 8 16 14 DNAArtificial Sequence Synthetic Construct 16 cgcttggcag tctc 14 17 14 DNAArtificial Sequence Synthetic Construct 17 cgcttggcag tctc 14 18 14 DNAArtificial Sequence Synthetic Construct 18 cgcttggcag tctc 14 19 16 DNAArtificial Sequence Synthetic Construct 19 tttaggattc gtgctc 16 20 12DNA Artificial Sequence Synthetic Construct 20 tcgtgctcat gg 12 21 8 DNAArtificial Sequence Synthetic Construct 21 gcgtttgc 8 22 16 DNAArtificial Sequence Synthetic Construct 22 cgctgcagat gcggtt 16 23 16DNA Artificial Sequence Synthetic Construct 23 ccgccggctc agtctt 16 2416 DNA Artificial Sequence Synthetic Construct 24 catcgtggcg gttagg 1625 15 DNA Artificial Sequence Synthetic Construct 25 tcgggtgagt ggtag 1526 16 DNA Artificial Sequence Synthetic Construct 26 cactcagtgc aactct16 27 16 DNA Artificial Sequence Synthetic Construct 27 cctccactcccgcctc 16 28 16 DNA Artificial Sequence Synthetic Construct 28catcgtggcg gttagg 16 29 16 DNA Artificial Sequence Synthetic Construct29 cactcagtgc aactct 16 30 16 DNA Artificial Sequence SyntheticConstruct 30 cctccactcc cgcctc 16 31 20 DNA Artificial SequenceSynthetic Construct 31 cagccatggt tccccccaac 20 32 15 DNA ArtificialSequence Synthetic Construct 32 gtgagggtct ctctc 15 33 15 DNA ArtificialSequence Synthetic Construct 33 gtgagggtct ctctc 15 34 15 DNA ArtificialSequence Synthetic Construct 34 caaatggttc tcgaa 15 35 15 DNA ArtificialSequence Synthetic Construct 35 caaatggttc tcgaa 15 36 15 DNA ArtificialSequence Synthetic Construct 36 acctgaggga gccag 15 37 15 DNA ArtificialSequence Synthetic Construct 37 acctgaggga gccag 15 38 15 DNA ArtificialSequence Synthetic Construct 38 ttggccacgt cctga 15 39 15 DNA ArtificialSequence Synthetic Construct 39 ttggccacgt cctga 15 40 15 DNA ArtificialSequence Synthetic Construct 40 tgcccgggaa aacgt 15 41 15 DNA ArtificialSequence Synthetic Construct 41 tgcccgggaa aacgt 15 42 15 DNA ArtificialSequence Synthetic Construct 42 cctcgtgcac gttct 15 43 15 DNA ArtificialSequence Synthetic Construct 43 cctcgtgcac gttct 15 44 15 DNA ArtificialSequence Synthetic Construct 44 tggatgtcga cctct 15 45 12 DNA ArtificialSequence Synthetic Construct 45 tttcgcgacc ca 12 46 12 DNA ArtificialSequence Synthetic Construct 46 tcgtgctcat gg 12 47 22 DNA ArtificialSequence Synthetic Construct 47 ntttaggatt cgtgctcatg gn 22 48 11 DNAArtificial Sequence Synthetic Construct 48 ttttcctctc n 11 49 10 DNAArtificial Sequence Synthetic Construct 49 aaaaggagag 10 50 10 DNAArtificial Sequence Synthetic Construct 50 gagaggaaaa 10 51 10 DNAArtificial Sequence Synthetic Construct 51 aaaagtagag 10 52 10 DNAArtificial Sequence Synthetic Construct 52 aaaaggtgag 10 53 10 DNAArtificial Sequence Synthetic Construct 53 gagatgaaaa 10 54 10 DNAArtificial Sequence Synthetic Construct 54 gagtggaaaa 10 55 10 DNAArtificial Sequence Synthetic Construct 55 aaaaaaaaaa 10 56 10 DNAArtificial Sequence Synthetic Construct 56 aaaaagaaaa 10 57 10 DNAArtificial Sequence Synthetic Construct 57 aaaaataaaa 10 58 10 DNAArtificial Sequence Synthetic Construct 58 aaaagaaaaa 10 59 10 DNAArtificial Sequence Synthetic Construct 59 aaaacaaaaa 10 60 10 DNAArtificial Sequence Synthetic Construct 60 aaaataaaaa 10

What is claimed is:
 1. A peptide nucleic acid having formula:

wherein: each L is independently selected from a group consisting ofnaturally-occurring nucleobases and non-naturally-occurring nucleobases,at least one of said L being a 2,6-diaminopurine nucleobase; each R^(7′)is independently hydrogen or C₁-C₈ alkylamine; R^(h) is OH, NH₂ orNHLysNH₂; R^(i) is H, COCH₃ or t-butoxycarbonyl; and n is an integerfrom 1 to
 30. 2. The peptide nucleic acid of claim 1 wherein said R^(7′)is C₃-C₆ alkylamine.
 3. The peptide nucleic acid of claim 2 wherein saidR^(7′) is C₄-C₅ alkylamine.
 4. The peptide nucleic acid of claim 3wherein said R^(7′) is butylamine.
 5. The peptide nucleic acid of claim1 wherein at least one of said R^(7′) is C₁-C₈ alkylamine.
 6. Thepeptide nucleic acid of claim 5 wherein at least one of said R^(7′) isC₃-C₆ alkylamine.
 7. The peptide nucleic acid of claim 6 wherein atleast one of said R^(7′) is C₄-C₅ alkylamine.
 8. The peptide nucleicacid of claim 7 wherein at least one of said R^(7′) is butylamine. 9.The peptide nucleic acid of claim 7 wherein substantially all of saidR^(7′) is butylamine.
 10. The peptide nucleic acid of claim 1 whereinthe carbon atom to which the substituent R^(7′) is attached isstereochemically enriched.
 11. The peptide nucleic acid of claim 10wherein said stereochemical enrichment is of the R configuration. 12.The peptide nucleic acid for claim 1 wherein said peptide nucleic acidis derived from an amino acid.
 13. The peptide nucleic acid for claim 12wherein said peptide nucleic acid is derived from D-lysine.
 14. Acompound having formula:

wherein: L is a 2,6-diaminopurine nucleobase; R^(7′) is hydrogen orC₁-C₈ alkylamine; E is COOH or an activated or protected derivativethereof; and Z is NH₂ or NHPg, where Pg is an amino-protecting group.15. The compound of claim 14 wherein said R^(7′) is C₃-C₆ alkylamine.16. The compound of claim 15 wherein said R^(7′) is C₄-C₅ alkylamine.17. The compound of claim 16 wherein said R^(7′) is butylamine.
 18. Thecompound of claim 14 wherein the carbon atom to which the substituentR^(7′) is attached is stereochemically enriched.
 19. The compound ofclaim 18 wherein said stereochemical enrichment is of the Rconfiguration.
 20. The compound of claim 14 wherein said compound isderived from an amino acid.
 21. The compound of claim 20 wherein saidcompound is derived from D-lysine.
 22. The compound of claim 14 whereinsaid Pg is t-butoxycarbonyl.
 23. A pharmaceutical composition comprisinga peptide nucleic acid according to claim 1 and at least onepharmaceutically effective carrier, binder, thickener, diluent, buffer,preservative, or surface active agent.